Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

BACKGROUND & AIMS: Liver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis. METHODS: Intracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor ß (TGFß) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model. RESULTS: In our study, we show that TGFß and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets' expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease. CONCLUSIONS: We provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.

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.

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.

ADAM17 cytoplasmic domain modulates Thioredoxin-1 conformation and activity.

The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.

Role of ADAM10 as a CD30 Sheddase in Classical Hodgkin Lymphoma.

Cancer cells generally recruit and influence non-malignant immune cells to support the tumor growth. Classical Hodgkin lymphoma (cHL) is a good example because the affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (H-RS) cells, which are supported by a massive infiltrate of lymphocytes, fibroblasts, and innate immune cells. The transmembrane receptor CD30, which is selectively expressed on the H-RS cells, plays an important role, not only in cell stimulation and intercellular communication but also in tumor diagnosis and targeted tumor therapy. Different protein processing pathways influence its functionality. Depending on the conditions, the receptor is internalized or released. The release of CD30 occurs either as an intact molecule, embedded in the membrane of extracellular vesicles (EVs), or as a cleaved soluble ectodomain (sCD30). CD30 cleavage is predominantly catalyzed by ADAM10. The enzyme is catalytically active in cells as well as in EVs and gradually releases sCD30. Because the circulation contains no CD30+ donor cells, this mechanism explains that the cleaved ectodomain represents the predominant form of CD30 in the plasma of cHL patients. CD30 processing might influence the impact of CD30 antibody-drug conjugates, such as Brentuximab Vedotin (BV). Whereas, ADAM10-degraded CD30 impedes the BV efficacy, tumor-derived EVs load bystander cells with CD30 and generate new targets among supporter cells. This crossfire effect might contribute to the enormous clinical impact of BV, whereas the ADAM10-dependent cleavage to the mild systemic off-target effects of the treatment with BV.

Exosome-dependent immune surveillance at the metastatic niche requires BAG6 and CBP/p300-dependent acetylation of p53.

Extracellular vesicles released by tumor cells contribute to the reprogramming of the tumor microenvironment and interfere with hallmarks of cancer including metastasis. Notably, melanoma cell-derived EVs are able to establish a pre-metastatic niche in distant organs, or on the contrary, exert anti-tumor activity. However, molecular insights into how vesicles are selectively packaged with cargo defining their specific functions remain elusive. Methods: Here, we investigated the role of the chaperone Bcl2-associated anthogene 6 (BAG6, synonym Bat3) for the formation of pro- and anti-tumor EVs. EVs collected from wildtype cells and BAG6-deficient cells were characterized by mass spectrometry and RNAseq. Their tumorigenic potential was analyzed using the B-16V transplantation mouse melanoma model. Results: We demonstrate that EVs from B-16V cells inhibit lung metastasis associated with the mobilization of Ly6Clow patrolling monocytes. The formation of these anti-tumor-EVs was dependent on acetylation of p53 by the BAG6/CBP/p300-acetylase complex, followed by recruitment of components of the endosomal sorting complexes required for transport (ESCRT) via a P(S/T)AP double motif of BAG6. Genetic ablation of BAG6 and disruption of this pathway led to the release of a distinct EV subtype, which failed to suppress metastasis but recruited tumor-promoting neutrophils to the pre-metastatic niche. Conclusion: We conclude that the BAG6/CBP/p300-p53 axis is a key pathway directing EV cargo loading and thus a potential novel microenvironmental therapeutic target.

Tumor-Derived Extracellular Vesicles Inhibit Natural Killer Cell Function in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Tumor-derived extracellular vesicles (EVs) induce pre-metastatic niche formation to promote metastasis. We isolated EVs from a highly-metastatic pancreatic cancer cell line and patient-derived primary cancer cells by ultracentrifugation. The protein content of EVs was analyzed by mass spectrometry. The effects of PDAC-derived EVs on natural kill (NK) cells were investigated by flow cytometry. The serum EVs' TGF-ß1 levels were quantified by ELISA. We found that integrins were enriched in PDAC-derived EVs. The expression of NKG2D, CD107a, TNF-α, and INF-γ in NK cells was significantly downregulated after co-culture with EVs. NK cells also exhibited decreased levels of CD71 and CD98, as well as impaired glucose uptake ability. In addition, NK cell cytotoxicity against pancreatic cancer stem cells was attenuated. Moreover, PDAC-derived EVs induced the phosphorylation of Smad2/3 in NK cells. Serum EVs' TGF-ß1 was significantly increased in PDAC patients. Our findings emphasize the immunosuppressive role of PDAC-derived EVs and provide new insights into our understanding of NK cell dysfunction regarding pre-metastatic niche formation in PDAC.

Extracellular vesicle measurements with nanoparticle tracking analysis - An accuracy and repeatability comparison between NanoSight NS300 and ZetaView.

The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern's NanoSight NS300 or Particle Metrix' ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0-2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7-8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9-36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0-4.7) than measurements by NanoSight NS300 (% CV range: 5.4-10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5-134.3) compared to ZetaView (% DTEM range: 111.8-205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8-6.7; ZetaView: 1.4-7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.

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 acids. 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 Reed-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 (α-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-1α, IL-6, and TNF-α), 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-α/NF-κB-signaling, as key factors directing the EV-dependent phenotype changes in fibroblasts. Confirming the in vitro data, we demonstrated that EVs promote α-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.

Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity.

AIMS: A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. RESULTS: Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1K72A and catalytic site mutant Trx-1C32/35S rescued ADAM17 activity, although the interaction with Trx-1C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1K72A mutant showed similar oxidant levels to Trx-1C32/35S, even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. INNOVATION: We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. CONCLUSION: This unexpected Trx-1K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 29, 717-734.

RIG-I activation induces the release of extracellular vesicles with antitumor activity.

Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate-RNA (3pRNA) triggers antitumor immunity predominantly via NK cell activation and direct apoptosis induction in tumor cells. However, how NK cells are mobilized to attack the tumor cells remains elusive. Here, we show that RIG-I activation induced the secretion of extracellular vesicles (EVs) from melanoma cells, which by themselves revealed antitumor activity in vitro and in vivo. RIG-I-induced EVs from melanoma cells exhibited an increased expression of the NKp30-ligand (BAG6, BAT3) on their surface triggering NK cell-mediated lysis of melanoma cells via activation of the cytotoxicity NK cell-receptor NKp30. Moreover, systemic administration of RIG-I-induced melanoma-EVs showed a potent antitumor activity in a melanoma mouse model in vivo. In conclusion, our data establish a new RIG-I-dependent pathway leading to NK cell-mediated tumor cell killing.

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.

An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities.

A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.3) × 10(4) M(-1) s(-1) and 2.4 (±0.3) × 10(3) M(-1) s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities.

CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin, in vitro.

The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells. Here, we investigated how CD30-positive EVs influence the efficacy of the CD30 antibody drug conjugate (ADC) Brentuximab Vedotin (SGN-35). The malignant cells and the EVs expressed the active sheddase ADAM10. ADAM10 cleaved and released the CD30 ectodomain (sCD30), causing a gradual depletion of SGN-35 binding sites on EVs and creating a soluble competitor of the ADC therapy. In a 3D semi-solid tumor microenvironment model, the EVs were retained in the matrix whereas sCD30 penetrated readily into the surrounding culture medium. This resulted in a lowered ratio of EV-associated CD30 (CD30EV) to sCD30 in the surrounding medium in comparison to non-embedded cultures. A low percentage of CD30EV was also detected in the plasma of cHL patients, supporting the clinical relevance of the model. The adherence of CD30EV but not sCD30 to CD30-/CD30L+ mast cells and eosinophils allowed the indirect binding of SGN-35. Moreover, SGN-35 damaged CD30-negative cells, provided they were loaded with CD30+ EVs.

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

Chronic lymphocytic 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 immunosurveillance 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.

Protrusion-guided extracellular vesicles mediate CD30 trans-signalling in the microenvironment of Hodgkin's lymphoma.

Classical Hodgkin's lymphoma (cHL)-affected lymphoid tissue contains only a few malignant Hodgkin and Reed-Sternberg (HRS) cells, which are disseminated within a massive infiltrate of reactive cells. In particular, the innate immune infiltrate is deemed to support tumour growth by direct cell-cell interaction. Since they are rarely found in close proximity to the malignant cells in situ, we investigated whether cHL-derived extracellular vesicles might substitute for a direct cell-cell contact. We studied the crosstalk of the transmembrane proteins CD30 and CD30 ligand (CD30L) because they are selectively expressed on HRS and innate immune cells, respectively. Here, we showed that HRS cells released both the ectodomain as a soluble molecule (sCD30) and the entire receptor on the surface of extracellular vesicles. The vesicle diameter was 40-800 nm, as determined by cryo- and immune electron microscopy. In addition to CD30, typical extracellular vesicle markers were detected by mass spectrometry and flow cytometry, including tetraspanins, flotillins, heat shock proteins and adhesion molecules. In contrast to sCD30, vesicles caused a CD30-dependent release of interleukin-8 in CD30L(+) eosinophil-like EoL-1 cells and primary granulocytes from healthy donors, underscoring the functionality of CD30 on vesicles. In extracellular matrix (ECM)-embedded culture of HRS cells, a network of actin and tubulin-based protrusions guided CD30(+) vesicles into the micro-environment. This network targeted CD30(+) vesicles towards distant immune cells and caused a robust polarization of CD30L. Confocal laser scanning microscopy of 30 µm sections showed a CD30 vesicle-containing network also in cHL-affected lymphoid tissue of both mixed-cellularity and nodular sclerosing subtypes. This network might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30-CD30L interaction in trans. The tubulin backbone of the network may provide a target for the therapy of cHL with antitubulin-based CD30 antibody constructs.

The bispecific immunoligand ULBP2-aCEA redirects natural killer cells to tumor cells and reveals potent anti-tumor activity against colon carcinoma.

NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody-derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re-directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell-mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti-tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD45+ immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti-tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.

The histone deacetylase inhibitor LBH589 (panobinostat) modulates the crosstalk of lymphocytes with Hodgkin lymphoma cell lines.

Epigenetic changes have been implicated in the malignant phenotype of Hodgkin Reed Sternberg (HRS) cells in Hodgkin lymphoma (HL), where HRS survival and proliferation depends on the microenvironment. The histone-deacetylase (HDAC) inhibitor LBH589 (panobinostat) showed clinical efficacy but its impact on the HRS microenvironment is unclear. Hence, we analysed the effects of LBH589 on lymphocytes and also potential combination therapies. In lymphocyte-target cell killing assays, LBH589-treatment triggered an enhanced lymphocyte-dependent lysis of HL cells despite of mild lymphocytopenic effects. In co-culture experiments of lymphocytes with HL cells, LBH589 suppressed the IFNgamma-release but increased the TNFalpha secretion. Recombinant TNFalpha boosted the lymphocyte-dependent lysis of HL target cells. In HL cell lines, LBH589 induced cell death, autophagy, and an increase of MICA/B that are ligands to natural killer cell receptors. The combination of LBH589 with Brentuximab Vedotin was inefficient due to down-regulation of CD30 as a target. Combination with gemcitabine revealed highly significant effects, suggesting a potential combination for future therapy. Based on these data we suggest that LBH589 favourably modulates the cytokine network and lymphocyte activity in the HL microenvironment.