Chemotherapy-induced release of ADAM17 bearing EV as a potential resistance mechanism in ovarian cancer.

Ovarian cancer (OvCa) is the gynaecological disorder with the poorest prognosis due to the fast development of chemoresistance. We sought to connect chemoresistance and cancer cell-derived extracellular vesicles (EV). The mechanisms of how chemoresistance is sustained by EV remained elusive. One potentially contributing factor is A Disintegrin and Metalloprotease 17 (ADAM17)-itself being able to promote chemoresistance and inducing tumour cell proliferation and survival via the Epidermal Growth Factor Receptor (EGFR) pathway by shedding several of its ligands including Amphiregulin (AREG). We now demonstrate that upon chemotherapeutic treatment, proteolytically active ADAM17 is released in association with EV from OvCa cells. In terms of function, we show that patient-derived EV induce AREG shedding and restore chemoresistance in ADAM17-deficient cells. Confirming that ADAM17-containing EV transmit chemoresistance in OvCa, we propose that ADAM17 levels (also on EV) might serve as an indicator for tumour progression and the chemosensitivity status of a given patient.

Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

Analysis of the Seasonal Fluctuation of γδ T Cells and Its Potential Relation with Vitamin D3.

In addition to its role in bone metabolism, vitamin D3 exerts immunomodulatory effects and has been proposed to contribute to seasonal variation of immune cells. This might be linked to higher vitamin D3 levels in summer than in winter due to differential sun exposure. γδ T cells comprise a numerically small subset of T cells in the blood, which contribute to anti-infective and antitumor immunity. We studied the seasonal fluctuation of γδ T cells, the possible influence of vitamin D3, and the effect of the active metabolite 1α,25(OH)2D3 on the in vitro activation of human γδ T cells. In a retrospective analysis with 2625 samples of random blood donors, we observed higher proportions of γδ T cells in winter when compared with summer. In a prospective study over one year with a small cohort of healthy adults who did or did not take oral vitamin D3 supplementation, higher proportions of γδ T cells were present in donors without oral vitamin D3 uptake, particularly in spring. However, γδ T cell frequency in blood did not directly correlate with serum levels of 25(OH)D3. The active metabolite 1α,25(OH)2D3 inhibited the in vitro activation of γδ T cells at the level of proliferation, cytotoxicity, and interferon-γ production. Our study reveals novel insights into the seasonal fluctuation of γδ T cells and the immunomodulatory effects of vitamin D3.

Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles.

Introduction: In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. Methods: We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Results: Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Conclusion: Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.

The Serine Protease CD26/DPP4 in Non-Transformed and Malignant T Cells.

CD26/Dipeptidylpeptidase 4 is a transmembrane serine protease that cleaves off N-terminal dipeptides. CD26/DPP4 is expressed on several immune cell types including T and NK cells, dendritic cells, and activated B cells. A catalytically active soluble form of CD26/DPP4 can be released from the plasma membrane. Given its wide array of substrates and interaction partners CD26/DPP4 has been implicated in numerous biological processes and effects can be dependent or independent of its enzymatic activity and are exerted by the transmembrane protein and/or the soluble form. CD26/DPP4 has been implicated in the modulation of T-cell activation and proliferation and CD26/DPP4-positive T cells are characterized by remarkable anti-tumor properties rendering them interesting candidates for T cell-based immunotherapies. Moreover, especially in cutaneous T-cell lymphoma CD26/DPP4 expression patterns emerged as an established marker for diagnosis and treatment monitoring. Surprisingly, besides a profound knowledge on substrates, interaction partners, and associated signal transduction pathways, the precise role of CD26/DPP4 for T cell-based immune responses is only partially understood.

CD30-Positive Extracellular Vesicles Enable the Targeting of CD30-Negative DLBCL Cells by the CD30 Antibody-Drug Conjugate Brentuximab Vedotin.

CD30, a member of the TNF receptor superfamily, is selectively expressed on a subset of activated lymphocytes and on malignant cells of certain lymphomas, such as classical Hodgkin Lymphoma (cHL), where it activates critical bystander cells in the tumor microenvironment. Therefore, it is not surprising that the CD30 antibody-drug conjugate Brentuximab Vedotin (BV) represents a powerful, FDA-approved treatment option for CD30+ hematological malignancies. However, BV also exerts a strong anti-cancer efficacy in many cases of diffuse large B cell lymphoma (DLBCL) with poor CD30 expression, even when lacking detectable CD30+ tumor cells. The mechanism remains enigmatic. Because CD30 is released on extracellular vesicles (EVs) from both, malignant and activated lymphocytes, we studied whether EV-associated CD30 might end up in CD30- tumor cells to provide binding sites for BV. Notably, CD30+ EVs bind to various DLBCL cell lines as well as to the FITC-labeled variant of the antibody-drug conjugate BV, thus potentially conferring the BV binding also to CD30- cells. Confocal microscopy and imaging cytometry studies revealed that BV binding and uptake depend on CD30+ EVs. Since BV is only toxic toward CD30- DLBCL cells when CD30+ EVs support its uptake, we conclude that EVs not only communicate within the tumor microenvironment but also influence cancer treatment. Ultimately, the CD30-based BV not only targets CD30+ tumor cell but also CD30- DLBCL cells in the presence of CD30+ EVs. Our study thus provides a feasible explanation for the clinical impact of BV in CD30- DLBCL and warrants confirming studies in animal models.

Intra- and Extracellular Effector Vesicles From Human T And NK Cells: Same-Same, but Different?

Cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells utilize an overlapping effector arsenal for the elimination of target cells. It was initially proposed that all cytotoxic effector proteins are stored in lysosome-related effector vesicles (LREV) termed "secretory lysosomes" as a common storage compartment and are only released into the immunological synapse formed between the effector and target cell. The analysis of enriched LREV, however, revealed an uneven distribution of individual effectors in morphologically distinct vesicular entities. Two major populations of LREV were distinguished based on their protein content and signal requirements for degranulation. Light vesicles carrying FasL and 15 kDa granulysin are released in a PKC-dependent and Ca2+-independent manner, whereas dense granules containing perforin, granzymes and 9 kDa granulysin require Ca2+-signaling as a hallmark of classical degranulation. Notably, both types of LREV do not only contain the mentioned cytolytic effectors, but also store and transport diverse other immunomodulatory proteins including MHC class I and II, costimulatory and adhesion molecules, enzymes (i.e. CD26/DPP4) or cytokines. Interestingly, the recent analyses of CTL- or NK cell-derived extracellular vesicles (EV) revealed the presence of a related mixture of proteins in microvesicles or exosomes that in fact resemble fingerprints of the cells of origin. This overlapping protein profile indicates a direct relation of intra- and extracellular vesicles. Since EV potentially also interact with cells at distant sites (apart from the IS), they might act as additional effector vesicles or intercellular communicators in a more systemic fashion.

Degranulation of human cytotoxic lymphocytes is a major source of proteolytically active soluble CD26/DPP4.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease detected on several immune cells and on epithelial cells of various organs. Besides the membrane-bound enzyme, a catalytically active soluble form (sCD26/DPP4) is detected in several body fluids. Both variants cleave off dipeptides from the N-termini of various chemokines, neuropeptides, and hormones. CD26/DPP4 plays a fundamental role in the regulation of blood glucose levels by inactivating insulinotropic incretins and CD26/DPP4 inhibitors are thus routinely used in diabetes mellitus type 2 therapy to improve glucose tolerance. Such inhibitors might also prevent the CD26/DPP4-mediated inactivation of the T-cell chemoattractant CXCL10 released by certain tumors and thus improve anti-tumor immunity and immunotherapy. Despite its implication in the regulation of many (patho-)physiological processes and its consideration as a biomarker and therapeutic target, the cellular source of sCD26/DPP4 remains highly debated and mechanisms of its release are so far unknown. In line with recent reports that activated T lymphocytes could be a major source of sCD26/DPP4, we now demonstrate that CD26/DPP4 is stored in secretory granules of several major human cytotoxic lymphocyte populations and co-localizes with effector proteins such as granzymes, perforin, and granulysin. Upon stimulation, vesicular CD26/DPP4 is rapidly translocated to the cell surface in a Ca2+-dependent manner. Importantly, activation-induced degranulation leads to a massive release of proteolytically active sCD26/DPP4. Since activated effector lymphocytes serve as a major source of sCD26/DPP4, these results might explain the observed disease-associated alterations of sCD26/DPP4 serum levels and also indicate a so far unknown role of CD26/DPP4 in lymphocyte-mediated cytotoxicity.

Granulysin species segregate to different lysosome-related effector vesicles (LREV) and get mobilized by either classical or non-classical degranulation.

Granulysin (GNLY) is a cationic antimicrobial, proinflammatory, and cytotoxic effector protein primarily expressed in human cytotoxic T and NK cells. Its two variants, the 15 kDa precursor and the mature 9 kDa protein processed by proteolysis, act on different microbes or infected and transformed target cells and utilize mechanistically different effector activities. In human peripheral blood lymphocytes of healthy individuals, both forms of GNLY are detected in TCR αß+ (CD4+ and CD8+) T cells, TCR γδ+ T cells, and CD3-CD56+ NK cells. In general, classical cytotoxic cells (i.e. CD8+ TCR αß+ T cells, TCR γδ+ T cells, and NK cells) contain effector proteins in higher abundance in more cells of the subset as compared to TCR αß+ CD4+ T cells. Imaging flow cytometry analyses demonstrate that the subcellular localization and internal pools of 9 kDa and 15 kDa GNLY are virtually non-overlapping. The 9 kDa form is enriched in dense granules that also contain granzymes (Grz) and carry CD107a, whereas 15 kDa GNLY is associated with CD107a-negative lysosome-related effector vesicles. We further demonstrate that 15 kDa GNLY serves as an additional indicator for non-classical, PKC-dependent degranulation while the liberation of granules containing 9 kDa GNLY requires calcium mobilization. Our studies provide a deeper insight into the subcellular localization and release mechanisms of the individual GNLY species. This information will not only be useful for the interpretation of GNLY-related pathophysiologies, but also for the development of therapeutic interventions employing distinct GNLY effector functions for microbial targeting or immunoregulation.

TGF-ß enhances the cytotoxic activity of Vδ2 T cells.

TGF-ß is a pleiotropic cytokine with multiple roles in immunity. Apart from its suppressive activity, TGF-ß is a driving cytokine in the differentiation of induced regulatory T cells (iTreg) but also in the polarization of interleukin-9 (IL-9) producing T helper 9 (Th9) T cells. Human Vδ2 expressing γδ T cells exert potent cytotoxicity towards a variety of solid tumor and leukemia/lymphoma target cells and thus are in the focus of current strategies to develop cell-based immunotherapies. Here we report that TGF-ß unexpectedly augments the cytotoxic effector activity of short-term expanded Vδ2 T cells when purified γδ T cells are activated with specific pyrophosphate antigens and IL-2 or IL-15 in the presence of TGF-ß. TGF-ß up-regulates the expression of CD54, CD103, interferon-γ, IL-9 and granzyme B in γδ T cells while CD56 and CD11a/CD18 are down-regulated. Moreover, we show that CD103 (αE/ß7 integrin) is recruited to the immunological synapse in γδ T cells. Increased cytotoxic activity of TGF-ß-exposed γδ T cells is reduced by anti-CD103 and further diminished upon additional anti-CD11a antibody treatment, pointing to a role of cellular adhesion in the enhanced cytolytic activity. Furthermore, magnetically sorted CD103-positive Vδ2 T cells exhibit superior cytolytic activity. In view of the importance of CD103 for tissue homing of lymphocytes, our results suggest that adoptive transfer of CD103-expressing Vδ2 T cells might favor their homing to solid tumors.

Mechanistic peculiarities of activation-induced mobilization of cytotoxic effector proteins in human T cells.

It is widely accepted that cytotoxic T and NK cells store effector proteins including granzymes, perforin and Fas ligand (FasL) in intracellular granules, often referred to as secretory lysosomes. Upon target cell encounter, these organelles are transported to the cytotoxic immunological synapse, where they fuse with the plasma membrane to release the soluble effector molecules and to expose transmembrane proteins including FasL on the cell surface. We previously described two distinct species of secretory vesicles in T and NK cells that differ in size, morphology and protein loading, most strikingly regarding FasL and granzyme B. We now show that the signal requirements for the mobilization of one or the other granule also differ substantially. We report that prestored FasL can be mobilized independent of extracellular Ca2+, whereas the surface exposure of lysosome-associated membrane proteins (Lamps; CD107a and CD63) and the release of granzyme B are calcium-dependent. The use of selective inhibitors of actin dynamics unequivocally points to different transport mechanisms for individual vesicles. While inhibitors of actin polymerization/dynamics inhibit the surface appearance of prestored FasL, they increase the activation-induced mobilization of CD107a, CD63 and granzyme B. In contrast, inhibition of the actin-based motor protein myosin 2a facilitates FasL-, but impairs CD107a-, CD63- and granzyme B mobilization. From our data, we conclude that distinct cytotoxic effector granules are differentially regulated with respect to signaling requirements and transport mechanisms. We suggest that a T cell might 'sense' which effector proteins it needs to mobilize in a given context, thereby increasing efficacy while minimizing collateral damage.

In-depth immunophenotyping of patients with glioblastoma multiforme: Impact of steroid treatment.

Despite aggressive treatment regimens based on surgery and radiochemotherapy, the prognosis of patients with grade IV glioblastoma multiforme (GBM) remains extremely poor, calling for alternative options such as immunotherapy. Immunological mechanisms including the Natural Killer Group 2 member D (NKG2D) receptor-ligand system play an important role in tumor immune surveillance and targeting the NKG2D system might be beneficial. However, before considering any kind of immunotherapy, a precise characterization of the immune system is important, particularly in GBM patients where conventional therapies with impact on the immune system are frequently co-administered. Here we performed an in-depth immunophenotyping of GBM patients and age-matched healthy controls and analyzed NKG2D ligand expression on primary GBM cells ex vivo. We report that GBM patients have a compromised innate immune system irrespective of steroid (dexamethasone) medication. However, dexamethasone drastically reduced the number of immune cells in the blood of GBM patients. Moreover, higher counts of immune cells influenced by dexamethasone like CD45+ lymphocytes and non-Vδ2 γδ T cells were associated with better overall survival. Higher levels of NKG2D ligands on primary GBM tumor cells were observed in patients who received radiochemotherapy, pointing towards increased immunogenic potential of GBM cells following standard radiochemotherapy. This study sheds light on how steroids and radiochemotherapy affect immune cell parameters of GBM patients, a pre-requisite for the development of new therapeutic strategies targeting the immune system in these patients.

Immunosurveillance by human γδ T lymphocytes: the emerging role of butyrophilins.

In contrast to conventional T lymphocytes, which carry an αß T-cell receptor and recognize antigens as peptides presented by major histocompatibility complex class I or class II molecules, human γδ T cells recognize different metabolites such as non-peptidic pyrophosphate molecules that are secreted by microbes or overproduced by tumor cells. Hence, γδ T cells play a role in immunosurveillance of infection and cellular transformation. Until recently, it has been unknown how the γδ T-cell receptor senses such pyrophosphates in the absence of known antigen-presenting molecules. Recent studies from several groups have identified a unique role of butyrophilin (BTN) protein family members in this process, notably of BTN3A1. BTNs are a large family of transmembrane proteins with diverse functions in lipid secretion and innate and adaptive immunity. Here we discuss current models of how BTN molecules regulate γδ T-cell activation. We also address the implications of these recent findings on the design of novel immunotherapeutic strategies based on the activation of γδ T cells.

Role of caspases in CD95-induced biphasic activation of acid sphingomyelinase.

Acid sphingomyelinase (A-SMase) plays an important role in the initiation of CD95 signaling by forming ceramide-enriched membrane domains that enable clustering and activation of the death receptors. In TNF-R1 and TRAIL-R1/R2 signaling, A-SMase also contributes to the lysosomal apoptosis pathway triggered by receptor internalization. Here, we investigated the molecular mechanism of CD95-mediated A-SMase activation, demonstrating that A-SMase is located in internalized CD95-receptosomes and is activated by the CD95/CD95L complex in a biphasic manner.Since several caspases have been described to be involved in the activation of A-SMase, we evaluated expression levels of caspase-8, caspase-7 and caspase-3 in CD95-receptosomes. The occurrence of cleaved caspase-8 correlated with the first peak of A-SMase activity and translocation of the A-SMase to the cell surface which could be blocked by the caspase-8 inhibitor IETD.Inhibition of CD95-internalization selectively reduced the second phase of A-SMase activity, suggesting a fusion between internalized CD95-receptosomes and an intracellular vesicular pool of A-SMase. Further analysis demonstrated that caspase-7 activity correlates with the second phase of the A-SMase activity, whereas active caspase-3 is present at early and late internalization time points. Blocking caspases-7/ -3 by DEVD reduced the second phase of A-SMase activation in CD95-receptosomes suggesting the potential role of caspase-7 or -3 for late A-SMase activation.In summary, we describe a biphasic A-SMase activation in CD95-receptosomes indicating (I.) a caspase-8 dependent translocation of A-SMase to plasma membrane and (II.) a caspase-7 and/or -3 dependent fusion of internalized CD95-receptosomes with intracellular A-SMase-containing vesicles.

TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death.

During apoptosis induction by TNF, the extrinsic and intrinsic apoptosis pathways converge at the lysosomal-mitochondrial interface. Earlier studies showed that the lysosomal aspartic protease Cathepsin D (CtsD) cleaves Bid to tBid, resulting in the amplification of the initial apoptotic cascade via mitochondrial outer membrane permeabilization (MOMP).The goal of this study was to identify further targets for CtsD that might be involved in activation upon death receptor ligation. Using a proteomics screen, we identified the heat shock protein 90 (HSP90) to be cleaved by CtsD after stimulation of U937 or other cell lines with TNF, FasL and TRAIL. HSP90 cleavage corresponded to apoptosis sensitivity of the cell lines to the different stimuli. After mutation of the cleavage site, HSP90 partially prevented apoptosis induction in U937 and Jurkat cells. Overexpression of the cleavage fragments in U937 and Jurkat cells showed no effect on apoptosis, excluding a direct pro-apoptotic function of these fragments. Pharmacological inhibition of HSP90 with 17AAG boosted ligand mediated apoptosis by enhancing Bid cleavage and caspase-9 activation. Together, we demonstrated that HSP90 plays an anti-apoptotic role in death receptor signalling and that CtsD-mediated cleavage of HSP90 sensitizes cells for apoptosis. These findings identify HSP90 as a potential target for cancer therapy in combination with death ligands (e.g. TNF or TRAIL).

Butyrophilin 3A/CD277-Dependent Activation of Human γδ T Cells: Accessory Cell Capacity of Distinct Leukocyte Populations.

Human Vγ9Vδ2 T cells recognize in a butyrophilin 3A/CD277-dependent way microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) or endogenous pyrophosphates (isopentenyl pyrophosphate [IPP]). Nitrogen-bisphosphonates such as zoledronic acid (ZOL) trigger selective γδ T cell activation because they stimulate IPP production in monocytes by inhibiting the mevalonate pathway downstream of IPP synthesis. We performed a comparative analysis of the capacity of purified monocytes, neutrophils, and CD4 T cells to serve as accessory cells for Vγ9Vδ2 T cell activation in response to three selective but mechanistically distinct stimuli (ZOL, HMBPP, agonistic anti-CD277 mAb). Only monocytes supported γδ T cell expansion in response to all three stimuli, whereas both neutrophils and CD4 T cells presented HMBPP but failed to induce γδ T cell expansion in the presence of ZOL or anti-CD277 mAb. Preincubation of accessory cells with the respective stimuli revealed potent γδ T cell-stimulating activity of ZOL- or anti-CD277 mAb-pretreated monocytes, but not neutrophils. In comparison with monocytes, ZOL-pretreated neutrophils produced little, if any, IPP and expressed much lower levels of farnesyl pyrophosphate synthase. Exogenous IL-18 enhanced the γδ T cell expansion with all three stimuli, remarkably also in response to CD4 T cells and neutrophils preincubated with anti-CD277 mAb or HMBPP. Our study uncovers unexpected differences between monocytes and neutrophils in their accessory function for human γδ T cells and underscores the important role of IL-18 in driving γδ T cell expansion. These results may have implications for the design of γδ T cell-based immunotherapeutic strategies.

NKG2D- and T-cell receptor-dependent lysis of malignant glioma cell lines by human γδ T cells: Modulation by temozolomide and A disintegrin and metalloproteases 10 and 17 inhibitors.

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) and UL-16 binding protein (ULBP) family members expressed on tumor cells with the corresponding NKG2D receptor triggers cytotoxic effector functions in NK cells and γδ T cells. However, as a mechanism of tumor immune escape, NKG2D ligands (NKG2DLs) can be released from the cell surface. In this study, we investigated the NKG2DL system in different human glioblastoma (GBM) cell lines, the most lethal brain tumor in adults. Flow cytometric analysis and ELISA revealed that despite the expression of various NKG2DLs only ULBP2 is released as a soluble protein via the proteolytic activity of "a disintegrin and metalloproteases" (ADAM) 10 and 17. Moreover, we report that temozolomide (TMZ), a chemotherapeutic agent in clinical use for the treatment of GBM, increases the cell surface expression of NKG2DLs and sensitizes GBM cells to γδ T cell-mediated lysis. Both NKG2D and the T-cell receptor (TCR) are involved. The cytotoxic activity of γδ T cells toward GBM cells is strongly enhanced in a TCR-dependent manner by stimulation with pyrophosphate antigens. These data clearly demonstrate the complexity of mechanisms regulating NKG2DL expression in GBM cells and further show that treatment with TMZ can increase the immunogenicity of GBM. Thus, TMZ might enhance the potential of the adoptive transfer of ex vivo expanded γδ T cells for the treatment of malignant glioblastoma.

Tetraspanin 8 is an interactor of the metalloprotease meprin ß within tetraspanin-enriched microdomains.

Meprin ß is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin ß cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid ß peptides and implicating a role of meprin ß in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin ß, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin ß. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin ß. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin ß. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin ß nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin ß at the cell surface with impact on certain proteolytic processes that have to be further identified.

Tetraspanin 8 is an interactor of the metalloprotease meprin ß within tetraspanin-enriched microdomains.

Meprin ß is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin ß cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid ß peptides and implicating a role of meprin ß in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin ß, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin ß. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin ß. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin ß. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin ß nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin ß at the cell surface with impact on certain proteolytic processes that have to be further identified.

Subcellular localization and activation of ADAM proteases in the context of FasL shedding in T lymphocytes.

The "A Disintegrin And Metalloproteinases" (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.