12033 research outputs found
Sort by
Dissecting mechanisms of CD8<sup>+</sup> T cell-mediated control of metastatic melanoma
No full textAlthough CD8+ T cells can play an anti-tumoural role in cancer, they often fail to do so, in part due to the acquisition of an ‘exhausted’ or ‘dysfunctional’ state. Different T cell subsets have been identified in this context, including stem-like precursor exhausted cells (Tpex) that help sustain responses. More differentiated exhausted subsets downstream of Tpex include both dysfunctional terminally exhausted (Ttex) and more cytotoxic CX3CR1+ subsets. Indeed, immune checkpoint blockade therapy appears to induce proliferation of Tpex cells and enhance differentiation of the effector-like CX3CR1+ subset. Despite this emerging understanding, the precise features of the CD8+ T cell response that enable successful tumour control remain incompletely defined. This is particularly the case for lymph node metastases, which are often the first site of metastasis in patients.
To address this, our group previously established a transplantable melanoma model whereby a proportion of mice develop metastases in the tumour-draining lymph node following curative-intent surgery of the orthotopic primary skin tumour. In this thesis, we further characterised this model and established situations of antigen-specific CD8+ T cell-driven control of lymph node metastases. We then interrogated the T cell response that was driving this control using cutting-edge techniques such as high-dimensional flow cytometry, single-cell RNA sequencing, and multiplex imaging. Firstly, we were able to maintain control of lymph node metastases over several weeks by transfer of in vitro-activated tumour-specific transgenic CD8+ T cells. In addition to ongoing control of metastases in some mice, others escaped therapy and rapidly grew out. We identified a less exhausted and more functionally activated T cell response in lesions under ongoing control relative to those that escaped therapy. These activated T cells preferentially localised to controlled lesions over the surrounding lymphoid tissue, and were associated with tumour cell cycle arrest, which may be contributing to the inhibition of metastasis outgrowth. Secondly, CD8+ T cell-driven spontaneous regression or eradication of lymph metastases was observed in Ifng–/– mice, but not their wild-type counterparts. Relative to wild-type mice, tumour-specific T cells in metastases from Ifng–/– mice showed greater differentiation, including an increase in the cytotoxic CX3CR1+ subset. Despite enhanced differentiation, Tpex cells were also maintained, which are known to be important for sustaining responses. In addition, T cells were more expanded in Ifng–/– mice, which also likely contributed to successful tumour control.
Overall, work in this thesis defined the spatial, phenotypic, and functional features of two different situations of CD8+ T cell-driven control of lymph node metastases. This provides novel insights into how successful CD8+ T cell responses to cancer are achieved and strong grounding for further work into unravelling the molecular mechanisms driving this. In turn, an improved understanding of these anti-tumoural responses could inform more effective and targeted clinical cancer immunotherapies in the future
Non-invasive physical plasma as an oncological therapy option: Modulation of cancer cell growth, motility, and metabolism without induction of cancer resistance factors
No full textBackground: Physical plasma, the fourth state of matter formed through gas ionization, has shown promise in various clinical applications, including wound healing and antimicrobial therapy. Recently, Non-invasive physical plasma (NIPP) selectively disrupts tumor cell proliferation and metabolism without inducing cytoprotective stress responses, positioning it as a promising adjunct in oncological therapies, though its underlying mechanisms remain insufficiently understood.
Methods: In this study, we investigated the effects of NIPP (Plasma Care device) on six tumor cell lines, ovarian (SKOV-3, OVCAR-3), prostate (LNCaP, PC-3), and breast (MCF-7, MDA-MB-231). Cell proliferation and migration were assessed using CASY analysis and scratch assays, while cytoskeletal integrity, heat shock protein (HSP) expression, and key metabolic indicators were evaluated through immunofluorescence, Western blotting, and biochemical assays.
Results: NIPP treatment significantly inhibited tumor cell proliferation and migration, disrupted cytoskeletal organization, and altered metabolic activity in a time-dependent manner. These effects were associated with increased intracellular reactive oxygen species (ROS), decreased mitochondrial membrane potential (MMP), enhanced glycolysis, and elevated lactate production. Notably, despite cellular stress, neither HSP expression nor superoxide dismutase (SOD) activity showed significant changes, suggesting a lack of classical stress-response activation.
Conclusions: Our findings indicate that NIPP selectively impairs tumor cell function by inducing oxidative stress and metabolic disruption, without triggering protective HSP-mediated resistance pathways commonly seen in radiotherapy and chemotherapy.These results highlight the therapeutic potential of NIPP, particularly via the Plasma Care device, as a novel anti-cancer strategy
Molecular pathomechanisms of immune-mediated epileptogenesis
No full textThis thesis explores the immune-mediated and molecular mechanisms underlying AE and epileptogenesis, with a particular emphasis on T cell-dependent neuroinflammation and the transcriptional dynamics across different stages of disease progression. AE, especially subtypes associated with intracellular autoantibodies, represents a contributor to the development of TLE. However, the precise immunopathological processes, including the initiation and maintenance of disease by T cells, remain incompletely understood. Moreover, beyond the context of AE, the broader mechanisms driving epileptogenesis involve complex, stage-specific molecular changes that are not yet fully characterized.
Although anti-GAD65 and anti-Drebrin-positive TLE patients display similar neuropathological features, they exhibit distinct immunopathogenic mechanisms. Single-nucleus RNA sequencing (snRNA-seq) of hippocampal tissue reveals that inflammatory signaling diverges between the subtypes: NF-κB pathway activation predominates in anti-GAD65-positive cases, while anti-Drebrin-positive patients show enrichment of FoxO-associated cascades. These differences might be reflected in distinct microglial phenotypes and correlate with varying clinical responses to therapy, underscoring the relevance of immune context in disease progression and therapeutic outcome.
Transcriptomic analyses in a T cell–mediated AE model further demonstrate that T cell activity alone, even in the absence of autoantibodies, is sufficient to trigger neuroinflammatory cascades, neuronal dysfunction, and early epileptogenic signatures. These findings underscore the central role of T cells and highlight shared downstream mechanisms between AE and broader forms of epilepsy. BBB dysfunction, which facilitates peripheral immune cell infiltration and disrupts the neurovascular niche, emerges as a critical factor in disease evolution. These targeted immune responses are accompanied by persistent BBB disruption, astrocytic paracrine dysfunction and leukocyte-driven inflammation. Moreover, cytotoxic T cell attack directed against GABAergic interneurons demonstrate to induce hallmark features of temporal lobe epilepsy TLE, including dentate gyrus granule cell dispersion and mossy fiber dysfunction. These findings show that T cell responses, shaped by the neuronal subtype they target, influence disease trajectories.
Furthermore, an in-depth analysis of early epileptogenesis using the pilocarpine-induced SE model of chronic TLE, highlights transcriptional cascades driven by immediate early genes and key regulators such as NFKB1, IRF8, and Spi110. These factors, activated shortly after seizure onset, initiate cell type–specific gene expression programs that promote sustained neuroinflammation and neuronal hyperexcitability. Despite differences in initiating insults and immune triggers, these molecular responses converge onto shared downstream pathways that define epileptogenic networks.
Taken together, this work identifies shared inflammatory and excitability-related mechanisms across AE and epileptogenesis, despite differing initiating triggers. Central elements include microglial activation, BBB disruption, and transcriptional programs driven by NF-κB and FoxO pathways. These findings underscore the potential of multi-targeted therapies such as GLP-1 receptor agonists and antioxidants to modulate both immune and oxidative stress responses. By identifying key molecular drivers, this thesis supports the development of precision medicine approaches to AE and epilepsy
Entwicklung von neuen Ti-Salen-basierten katalytischen Systemen
No full text(noch nicht zugänglich / not yet accessible
Investigating the Role of Nucleic Acid Sensing in the Pathogenesis of Chronic Viral Infection-Induced Chronic Kidney Disease and Autoimmunity
No full textPatients with chronic HIV infections, are at increased risk of developing comorbidities, with chronic kidney disease (CKD) such as immune complex glomerulonephritis (ICGN) being particularly common. Diagnosis of ICGN typically involves kidney biopsies, revealing significant deposition of immunoglobulins (Igs) and complements in the kidney glomeruli. The molecular mechanisms underlying HIV-associated CKD remain poorly understood, partly due to the lack of appropriate animal models.
Our clinical study identified increased levels of autoantibodies and circulating free DNA in the HIV patients with CKD compared to those without CKD, along with a strong IFN-I response. These pathological features can be reproduced in a mouse model of neoLCMV infection. In this model, neonates of C57BL6/J mice infected with WE-LCMV develop persistent infection and displayed strong deposition of Igs and complements in the kidney glomeruli, similar to what is observed in CKD positive HIV patients. This makes the neoLCMV mouse model a valuable tool to study RNA-viral infection-associated CKD.
Moreover, neoLCMV infection seems to drive the immune response towards an extrafollicular (ExFO) pathway of B cell activation. In the absence of TLR7, CKD in neoLCMV-infected mice is alleviated, accompanied with decreased autoantibodies production and a reduction in CXCR3+MHCIIhi ExFO plasma cells and ICOShiCXCR4+ ExFO T helper cells (ExFO-Th).
Disease amelioration was also obtained by specifically deleting TLR7 signaling in B cells using TLR7flxCD19cre/WT mice. These mice exhibited reduced B cell differentiation and altered ExFO-Th to Treg ratios, suggesting a novel intrinsic role of TLR7 in the B and T cell activation beyond its function as an RNA sensor. Our data also showed, that IL-10 and IL-6 production, which are strongly expressed in neoLCMV mice developing CKD, were significantly reduced in the absence of TLR7 signaling in B cells. Overall, these findings highlight the crucial role of TLR7 signaling in the CKD pathogenesis during chronic RNA viral infections and suggest that targeting TLR7 could be a potential strategy for treating of preventing HIV-associated CKD
Impact of G-Quadruplexes on DNA damage and repair supported by the protein Zuo1 in <em>Saccharomyces cerevisiae</em>
No full textThe secondary structures known as G-Quadruplexes (G4s) play crucial roles in regulating biological processes such as replication, transcription, and DNA repair by folding and unfolding within specific regions of DNA. Recently, non-canonical, less stable G4s have gained attention due to their high flexibility and potential to fine-tune critical processes like genome stability. The newly identified G4-binding protein Zuo1 is essential in regulating the formation of these structures. This study explores whether Zuo1 regulates the formation of non-canonical G4s and examines the function of the Zuo1-G4 interaction in DNA damage and repair, particularly in chromatin and epigenetic maintenance in Saccharomyces cerevisiae.
I discovered that the C-terminus of Zuo1 binds, stabilizes, and accelerates the formation of the yeast-specific G-quadruplex located on chromosome IX. This binding effect is influenced by the different topologies of G4IX, which depends on the salt environment. Additionally, Zuo1 shows a preferential binding to non-canonical G4s genome-wide, located primarily in promoter and telomeric regions. This indicates that Zuo1 may function as a transcription factor, altering the chromatin landscape by binding to G4s and interacting with chromatin remodelers and other transcription factors. Upon UV exposure, G4 formation protects DNA from damage. In Zuo1-deficient cells, where G4s are less stabilized, cyclobutene pyrimidine dimers accumulate as DNA damage products, and DNA repair is inefficient. However, stabilizing G4s with a G4-stabilizing compound rescues this phenotype. The absence of histone modifications, DNA polymerase binding, and changes in nucleosome organization in cells lacking Zuo1 underscore the critical role of G4 stabilization in chromatin regulation and epigenetics, affecting processes such as replication and DNA repair under both unchallenged and DNA damage conditions.
Understanding proteins that fine-tune processes by regulating non-canonical G4s could present new therapeutic opportunities. Many G4-binding compounds developed for cancer therapy have struggled to target specific G4s effectively, yet the C-terminus of Zuo1 exhibits selective binding to this subset of non-canonical G4 structures. Further characterization of the processes involving Zuo1-G4 interaction is necessary to comprehend their mechanisms and the specific regulation of cellular processes like DNA damage and repair, potentially through epigenetic regulation
Large-scale omics-based assessments of aging trajectories in flies and mice
No full textThe significant increase in life expectancy over the past two centuries can be attributed to advancements in public health, including improved sanitation, access to clean drinking water, widespread immunization, and the development of antibiotics. However, the expansion of human lifespan has led to a rise in chronic diseases typically linked to advanced age, thus shifting the burden of illness to later stages of life. The hallmarks of aging encompass biological processes such as genomic instability, telomere attrition, epigenetic alterations, and several factors. Conventional methods of measuring aging, like relying on lifespan as a proxy for aging, are not sufficient to sufficiently address the complex nature of aging. Therefore, it is necessary to adopt a more comprehensive approach that takes into account the many facets of aging and age-dependent change in physiological function. This thesis utilizes proteomics and transcriptomics to uncover a specific set of age-sensitive biomarkers and investigate their dynamic expression patterns. The research is organized into three interrelated chapters: the first chapter examines the proteomic landscape of normal aging across mouse tissues, the second chapter investigates genetic expression patterns associated with the stabilization of mortality in late life, and the third chapter explores the effects of environmental temperature on aging.
Our findings reveal distinct age-dependent proteomic changes across various organs, emphasizing the dynamic and organ-specific nature of aging. The identification of ASPs and their unique expression patterns provides deeper insights into the molecular mechanisms of aging, with implications for potential biomarkers of age-related diseases. Additionally, the study of Drosophila melanogaster demonstrates a stabilization phase in gene expression associated with a mortality plateau, suggesting biological mechanisms that limit further aging-related deterioration. This phenomenon is further validated by the evaluation of GFP reporter line-Drosomycin as one of the ASGs, offering a deeper perspective on plateau phase at the individual level. To ensure this is a convincing finding, more investigations into the GFP reporter lines are ongoing. Lastly, our investigation into the effects of ambient temperature on lifespan highlights the enduring influence of environmental factors, with transient exposure to low temperature extending lifespan and improving motor performance in later life stages. As previously said, lifespan is not an accurate proxy of aging due to its susceptibility to age-related pathologies like cancer. Proteome analysis will be conducted to evaluate the effect of transient exposure to low temperature on aging extending beyond the lifespan. In summary, this thesis improves our comprehension of age-dependent changes in the multi-omics profiles over time, shedding light on the nature of the aging progress. These findings provide a solid foundation for future studies that aim to understand a wide range of molecular processes involved in aging and find ways to support healthy aging and examine the effect of potential anti-aging therapies on age-related illnesses
The role of the innate immune system in acute Graft-versus-Host disease
No full textAcute Graft-versus-Host disease (aGvHD) remains a potentially lethal complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and an obstacle to curing blood cancer. A persistent challenge in finding new treatment options is the ambiguous role of allo-reactive donor T cells, which mediate both graft-versus-host but also graft-versus-leukemia (GvL) immunity. This thesis explores how the innate immune system contributes to aGvHD development and could be exploited for treatment.
Here, we establish a common MHC-mismatched mouse model for aGvHD (C57BL6/N → BALB/c) using x-ray total body irradiation (TBI). Although considerably lower than previously reported target dosages, TBI with 5 Gy provides sufficient myeloablation and facilitates donor engraftment. Allo-HSCT with bone marrow and T cells results in non-lethal radiation sickness and subsequent clinical manifestation of aGvHD.
Using this model, we investigate the potential of myeloperoxidase (MPO) inhibition as a means to target neutrophils. Our data show that preemptive inhibition of MPO through treatment with ABAH prior to the conditioning improves aGvHD severity and lethality. ABAH-treated mice present with decreased serum levels of pro-inflammatory cytokines (IL-1, TNFα, CXCL1), lipocalin-2, and liver enzymes shortly after allo-HSCT, indicating ameliorated early inflammation and tissue damage. Treatment with ABAH also reduced T cell infiltration and inflammation in liver and small intestine on day 21 after allo-HSCT. Interestingly, GvL activity remained unaffected by MPO inhibition, suggesting that the GvL effect is at least partially independent of the early events leading to aGvHD. Furthermore, ABAH treatment did not result in impaired bacterial clearance during urinary tract infection (UTI) with E. coli.
We also investigated whether eosinophils contribute to aGvHD development but found that neither adoptive transfer nor antibody-mediated depletion of eosinophils in recipient mice altered aGvHD severity and lethality.
In conclusion, our study provides evidence that neutrophil-derived MPO exacerbates conditioning-induced inflammation and tissue damage, which subsequently drives aGvHD pathogenesis, but is dispensable for GvL activity. Therefore, targeting the innate immune system, for example by inhibition of MPO, presents a promising prophylactic strategy for the prevention of aGvHD without increased risks of relapse or infection
Targeting tumor cells with cytokine-induced killer cells and bifunctional peptides
No full textAntimicrobial peptides have gained much attention in clinical research due to their extensive possibilities of application beyond antimicrobial use. The modification of antimicrobial peptides enables the peptides to target particular cancer cells, improving the specificity and efficiency of the treatment. In this study, TP2-D-Tox, a derivate of TP-D-Tox, demonstrated a superior anti-tumor activity towards renal carcinoma, Caki-2 and breast carcinoma, SK-BR-3. TP-and TP2-D-Tox were shown to penetrate the cells via clathrin-mediated endocytosis, triggered by binding to S100A9 and the subunits of non-muscle myosin IIa, including MYL6 in Caki-2 and MYL12A, MYL12B in SK-BR-3. TP2-D-Tox interaction with HSPB1 was observed to protect the intracellular peptide against the immediate proteolytic inactivation. Higher affinity of TP2-D-Tox towards these ligand proteins might have contributed to their larger therapeutic indices compared to TP-D-Tox.
Despite the occurrence of similar stress responses, our investigation revealed that TP- and TP2-D-Tox induced a distinctly regulated cell death in Caki-2 and SK-BR-3 cells in caspase-independent manner. The intracellular presence of the peptides evoked mitochondrial dysfunctions, elevation of cytosolic calcium, generation of reactive oxygen species and formation of MLKL oligomers in the plasma membrane preceding the necroptotic cell death in Caki-2 or necrotic cell death in SK-BR-3.
TP2-D-Tox superiority in triggering cell death represented a promising approach to bypass the caspase-dependent apoptosis-resistance issue impairing therapeutic responses of many cancer treatments. Combining TP2-D-Tox and CIK cells as cancer therapy may bypass several cancer resistance phenotypes forfeiting the neoevolutionary advantages.
The TP2-D-Tox-CIK cells combination cancer therapy revealed a greater efficacy in decimating Caki-2 and SK-BR-3 cells. Although the combination therapy failed to show therapeutic benefits over TP2-D-Tox monotreatment against Caki-2 monolayer, their antitumor activity demonstrated synergistic interactions in combating Caki-2 spheroids and SK-BR-3 in both models. The TP2-D-Tox-CIK cells combination therapy might have bypassed the limitation of the classical synergistic combination, which promotes the competitive advantage of cancer cells to developed single-treatment resistance annulling the overall efficacy of the combination therapy
Deciphering the regulation of tissue adaptation of regulatory T cells
No full textRegulatory T (Treg) cells are specialised immune cells of the CD4+ T cell lineage that are indispensable for the maintenance of immune homeostasis and prevention of immune-mediated pathology. Most Treg cells develop in the thymus and undergo a distinct differentiation program in the periphery, which is essential for the acquisition of a fully suppressive effector phenotype. This process endows Treg cells with the ability to migrate to multiple non-lymphoid tissues, where they mature and acquire tissue-specific features. Notably, tissue-resident Treg cells perform multiple non-immune functions, including the maintenance of tissue integrity and repair, as well as the regulation of systemic metabolism. In this study, we investigated the molecular control of tissue-specific effector Treg cell adaptation, focusing on two non-lymphoid tissues, the central nervous system (CNS) and adipose tissue. The CNS, composed of the brain and spinal cord, is responsible for reception, processing, and reaction to sensory stimuli. Recent studies have revealed the presence of immune cells, including Treg cells, in the CNS. However, the role of Treg cells in chronic CNS diseases, such as Multiple Sclerosis, remains poorly understood. In this study, we employed a mouse model of experimental autoimmune encephalomyelitis, recapitulating human Multiple Sclerosis, to investigate the effects of hypoxia-inducible factor 1-alpha (Hif1a) on Treg cells during disease progression. Hif1a, a transcription factor induced by hypoxia and inflammation, is elevated in Multiple Sclerosis patients. Independently, it has been shown to attenuate Treg cell development and function. My findings indicate that Treg cells that lack Hif1a switch to mitochondrial respiration at sites of inflammation. This change may enhance their effector function, which could have contributed to the alleviation of disease progression in female mice. Visceral adipose tissue (VAT) functions as an endocrine organ vital for metabolic homeostasis and energy storage. However, excessive accumulation of white adipose tissue due to aging or diet triggers tissue inflammation, increasing the risk of metabolic disorders, such as type 2 diabetes. Given the global obesity rise, understanding the interaction of the immune system with VAT is essential. Treg cells are critical for dampening VAT inflammation preserving tissue homeostasis and systemic metabolism. Research, including our own, indicates that a heterogeneous population of Treg cells accumulates in the VAT in a sex-dependent manner under steady-state conditions. These Treg cells can be categorised into two main clusters based on their expression of the interleukin (IL)-33 receptor ST2 and the chemokine receptor CXCR3. In male animals, prototypical ST2+ Treg cells, which depend on the transcription factors PPAR? and GATA3, are predominant. Conversely, CXCR3+ Treg cells, which rely on the transcription factor T-bet, are enriched in female mice. In addition to distinct phenotypical and functional characteristics, this study revealed differences in the metabolic requirements of these Treg cell populations showing elevated usage of amino acid metabolism in ST2+ Treg cells, while CXCR3+ Treg cells utilised mitochondrial respiration to maintain their high proliferative capacity. Furthermore, this study utilised novel transgenic mouse models, dietary conditions, and in vitro assays to investigate VAT microenvironmental signals that shape Treg cell differentiation, heterogeneity, and function. Our results identified a pivotal role for transforming growth factor-beta (TGF-ß) in maintaining VAT Treg cell homeostasis. Ablation of TGF-ß signalling in Treg cells favoured the expansion of CXCR3+ VAT Treg cells while reducing their overall sensitivity to IL-33 by the loss of ST2 expression. Phenotypic and functional changes in Treg cells in turn reshaped the cellular composition of VAT, creating an anti-inflammatory environment that prevented fat accumulation, even under high fat diet. Together, these results highlight a novel pathway by which TGF-ß steers VAT Treg cell differentiation and function, thereby contributing to the maintenance of VAT immune homeostasis and tissue morphology. Therefore, manipulation of Treg cells via TGF-ß may constitute a new, promising therapeutic target for metabolic diseases