The Role of CD95 Glycosylation in CD95 Signaling

The CD95 (APO-1/Fas)-mediated apoptotic pathway is one of the well-studied death receptor pathways. CD95 is a highly glycosylated type I transmembrane protein and has a molecular mass of 37.5 kDa. The N-terminal domain of CD95 contains two putative N-linked glycosylation sites at N118 and N136 and posttranslational glycosylation leads to an increase of molecular mass of CD95 to 45–52 kDa. Upon stimulation of CD95, either with its natural ligand CD95L or with agonistic antibodies such as anti-APO-1, a Death-Inducing Signaling Complex (DISC) is formed. The DISC consists of oligomerized, most probably trimerized, CD95, the DD-containing adaptor molecule Fas associated death domain containing protein (FADD), procaspase-8, procaspase-10 and cellular FLICE inhibitory protein (c-FLIP). The interactions between the molecules in the DISC are based on homophilic contacts. The death domain (DD) of CD95 interacts with the DD of FADD while the death effector domain (DED) of FADD interacts with the N-terminal tandem DED of procaspase-8. Binding of procaspase-8 to the DISC results in its autocatalytic cleavage with the formation of the mature caspase-8 which is released into the cytosol to propagate the apoptotic signal. The data presented here show that N-glycosylation of CD95 influences death-inducing signaling complex (DISC) formation and procaspase-8 activation at the CD95 DISC. Treatment with the chemical inhibitors of N-glycosylation tunicamycin and Deoxymannojirimycin (DMM), neuraminidase from Vibrio cholerae and generation of CD95 glycosylation mutants at N136 and N118 show that N deglycosylation of CD95 results in the reduction of caspase-8 processing at the DISC. Modelling of the core CD95 DISC structure and the structure of the CD95 DISC network on the membrane indicated that the glycan attached to N118 could be important for the contacts between neighboring DISCs forming a network. A glycan moiety attached to N136 of CD95 could be important for the complex formation and/or stability as it is located in close proximity to one of the CD95L molecules. The present study shows that the CD95 glycostructure can contribute to the amount of generated caspase-8, which defines cell death initiation. This regulation might be important for cancer cells when a subtle difference in the amount of activated caspase-8 regulates life or death of the cell

Modulation of the CD95-Induced Apoptosis: The Role of CD95 N-Glycosylation

Protein modifications of death receptor pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of TRAIL-receptor (R) is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. In this study we ask whether and how glycosylation of CD95 (Fas/APO-1), another death receptor, influences DISC formation and procaspase-8 activation at the CD95 DISC and thereby the onset of apoptosis. We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on the signal transduction: in silico modeling of CD95 DISC, generation of CD95 glycosylation mutants (at N136 and N118), modulation of N-glycosylation by deoxymannojirimycin (DMM) and sialidase from Vibrio cholerae (VCN). We demonstrate that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of the procaspase-8 activation at the DISC. These findings are important for the better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

DNA damage response and evasion from immunosurveillance in CLL: new options for NK cell-based immunotherapies

Chronic lyrnphocytic leukemia (CLL) is the most prominent B cell malignancy among adults in the Western world and characterized by a clonal expansion of B cells. The patients suffer from severe immune defects resulting in increased susceptibility to infections and failure to generate an antitumor immune response. Defects in both, DNA damage response (DDR) pathway and crosstalk with the tissue microenvironment have been reported to play a crucial role for the survival of CLL cells, therapy resistance and impaired immune response. To this end, major advances over the past years have highlighted several T cell immune evasion mechanisms in CLL. Here, we discuss the consequences of an impaired DDR pathway for detection and elimination of CLL cells by natural killer (NK) cells. NK cells are considered to be a major component of the irnmunosurveillance in leukemia but NK cell activity is impaired in CLL. Restoration of NK cell activity using immunoligands and immunoconstructs in combination with the conventional chemotherapy may provide a future perspective for CLL treatment

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia that affects B lymphocytes in adults. Natural killer (NK) cells in CLL patients are intrinsically potent but display poor in situ effector functions. NKG2D is an activating receptor found on NK and CD8(+) T cells and plays a role in immunosurveillance of CLL. In this study, we developed mono- and dual-targeting triplebodies utilizing a natural ligand for human NKG2D receptor (ULBP2) to retarget NK cells against tumor cells. Triplebodies in both formats showed better ability to induce NK-cell-dependent killing of target cells compared to bispecific counterparts. A mono-targeting triplebody ULBP2-aCD19-aCD19 successfully triggered NK cell effector functions against CLL cell line MEC1 and primary tumor cells in allogenic and autologous settings. Additionally, a dual-targeting triplebody ULBP2-aCD19-aCD33 specific for two distinct tumor-associated antigens was developed to target antigen loss variants, such as mixed lineage leukemia (MLL). Of note, this triplebody exhibited cytotoxic activity against CD19/CD33 double positive cells and retained its binding features even in the absence of one of the tumor antigens. Further, ULBP2-aCD19-aCD19 showed significant in vivo activity in immune-deficient (NSG) mouse model transplanted with CLL cell line as target cells and human immune cells as an effector population providing a proof-of-principle for this therapeutic concept

Hodgkin Lymphoma-Derived Extracellular Vesicles Change the Secretome of Fibroblasts Toward a CAF Phenotype

Secretion of extracellular vesicles (EVs) is a ubiquitous mechanism of intercellular communication based on the exchange of effector molecules, such as growth factors, cytokines, and nucleic adds. Recent studies identified tumor-derived EVs as central players in tumor progression and the establishment of the tumor microenvironment (TME). However, studies on EVs from classical Hodgkin lymphoma (cHL) are limited. The growth of malignant Hodgkin and Rood Sternberg (HRS) cells depends on the TME, which is actively shaped by a complex interaction of HRS cells and stromal cells, such as fibroblasts and immune cells. HRS cells secrete cytokines and angiogenic factors thus recruiting and inducing the proliferation of surrounding cells to finally deploy an immunosuppressive TME. In this study, we aimed to investigate the role of tumor cell-derived EVs within this complex scenario. We observed that EVs collected from Hodgkin lymphoma (HL) cells were internalized by fibroblasts and triggered their migration capacity. EV-treated fibroblasts were characterized by an inflammatory phenotype and an upregulation of alpha-smooth muscle actin (alpha-SMA), a marker of cancer-associated fibroblasts. Analysis of the secretome of EV-treated fibroblast revealed an enhanced release of pro-inflammatory cytokines (e.g., IL-1 alpha, IL-6, and TNF-alpha), growth factors (G-CSF and GM-CSF), and pro-angiogenic factors such as VEGF. These soluble factors are known to promote HL progression. In line, ingenuity pathway analysis identified inflammatory pathways, including TNF-alpha/NF-kappa B signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote alpha-SMA expression in fibroblasts and the expression of proangiogenic factors using a xenograft HL model. Collectively, we demonstrate that HL EVs alter the phenotype of fibroblasts to support tumor growth, and thus shed light on the role of EVs for the establishment of the tumor-promoting TME in HL