Apoptosis In Target Cells Research Articles

Use of real-time live-cell analysis with enhanced data analytics to quantify immune cell tumor killing

Abstract To develop new therapeutics, researchers are exploring the role of the immune system in defending the body against tumors. Modelling induced malignant cell death in vitro is of paramount importance. Tumor and immune cell co-cultures were created in 96 well plates and using live-cell analysis, various parameters of tumor killing were quantified in real-time. Red nuclear labeled target cells and various densities of pre-activated PBMCs (α-CD3/IL2, 4 d) were seeded in combination with IncuCyte Annexin V green apoptosis detection reagent. Images were acquired every 2 h for 3 d using IncuCyte. Analysis of the fluorescence images provides measurement of target cell number and apoptosis. Enhancement of the phase contrast image analytics enabled single cell segmentation, permitting determination of effector cell parameters; cell number, shape and, using fluorescently labeled surface marker antibodies, protein expression levels. In addition, studies into spatial interactions of target and effector cells were conducted. To exemplify how these new analytical features can be used to investigate the biology of tumor cell killing, studies of a α-hCD3xCD19 bi-specific T-cell engager antibody induced cytotoxicity were performed. Further characterization of effects on cell cycle during target cell death and use of more advanced 3D models of immune cell killing were also assessed, demonstrating the flexibility of live-cell analysis as a powerful tool for analyzing immune cell killing. Advances in data analytics has enabled the multiplexing of target cell quantification alongside the interrogation of effector cell properties in live cells. The added insight gained from these approaches will hopefully lead to improved immuno-therapeutics.

Crystal structure of rice defensin OsAFP1 and molecular insight into lipid-binding

Defensins are antibacterial peptides that function in the innate immune system. OsAFP1, a defensin identified from Oryza sativa (rice), exhibits antimicrobial activity against rice pathogens. Intriguingly, OsAFP1 was also shown to demonstrate potent antifungal activity against the human pathogenic fungus Candida albicans by inducing apoptosis in target cells, suggesting that OsAFP1 represents a potential new antibiotic candidate; however, further analyses, particularly at the structural level, are required to elucidate the mechanistic underpinnings of OsAFP1 antifungal activity. Here, we determined the three-dimensional structure of OsAFP1 using X-ray crystallography. OsAFP1 features the cysteine-stabilized αβ structure highly conserved in plant defensins and presents a dimeric structure that appears necessary for antifungal activity. Superimposition of the OsAFP1 structure with that of Nicotiana alata NaD1 complexed with phosphatidic acid indicated that the target molecule is likely trapped between the S2-S3 loops of each OsAFP1 dimer. In lipid-binding analyses performed using nitrocellulose membranes immobilized with various membrane lipid components, OsAFP1 was found to bind to phosphatidylinositols (PIPs) harboring phosphate groups, particularly PI(3)P. These results indicate that OsAFP1 exerts antifungal activity by binding to PI(3)P contained in the C.albicans cell membrane, thereby applying cellular stress and inducing apoptosis. Furthermore, the OsAFP1 structure and site-specific-mutation analyses revealed that Arg1, His2, Leu4, Arg9, and Phe10 play critical roles in OsAFP1 dimer formation. Thus, our study provides novel insights into the antifungal mechanism of OsAFP1.

Characterization of Human NK Cell-Derived Exosomes: Role of DNAM1 Receptor In Exosome-Mediated Cytotoxicity Against Tumor.

Despite the pivotal role of natural killer (NK) cells in defenses against tumors, their exploitation in cancer treatment is still limited due to their reduced ability to reaching tumor sites and the inhibitory effects of tumor microenvironment (TME) on their function. In this study, we have characterized the exosomes from IL2- or IL15-cultured human NK cells. Both cytokines induced comparable amounts of exosomes with similar cargo composition. Analysis of molecules contained within or exposed at the exosome surface, allowed the identification of molecules playing important roles in the NK cell function including IFN-γ, Lymphocyte Function-Associated Antigen (LFA-1), DNAX Accessory Molecule-1 (DNAM1) and Programmed Cell Death Protein (PD-1). Importantly, we show that DNAM1 is involved in exosome-mediated cytotoxicity as revealed by experiments using blocking antibodies to DNAM1 or DNAM1 ligands. In addition, antibody-mediated inhibition of exosome cytotoxicity results in a delay in target cell apoptosis. We also provide evidence that NK-exosomes may exert their cytolytic activity after short time interval and even at low concentrations. Regarding their possible use in immunotherapy, NK exosomes, detectable in peripheral blood, can diffuse into tissues and exert their cytolytic effect at tumor sites. This property offers a clue to integrate cancer treatments with NK exosomes.

Spontaneous and activation-induced apoptosis of peripheral blood mononuclear cells in the pathogenesis of type 1 diabetes mellitus

Apoptosis is the leading mechanism of pancreatic β-cell destruction in type 1 diabetes mellitus (T1DM). Assessment of activation apoptosis in response to stimulation with mitogen or a specific antigen is considered more significant when studying apoptosis markers in peripheral blood mononuclear cells to clarify the role of programmed cell death in pathogenesis of various diseases, since the role of apoptosis in immune response is increased in activated cells. It has been established that the stimuli that activate resting Tlymphocytes initiate apoptotic death of activated T lymphocytes. Therefore, detection of spontaneous apoptosis only is not very informative. In addition, determination of cell sensitivity to apoptosis induction makes it possible to identify relations of pathological process to the enhancement or weakening of this sensitivity. The key point in the T1DM initiation is apoptosis resistance of activated autoreactive T lymphocytes, that migrate from bloodstream to the pancreas and take an active part in destruction of the pancreatic insular structures. Despite long studies of T1DM pathogenesis, the exact causes of resistance of effector T cell clones to apoptosis remain unclear. There are no facts that answer the question: to what extent is the ability of T lymphocytes of peripheral blood to enter into apoptosis associated with severity and duration of the disease. In this regard, the aim of the study was to evaluate the effectiveness of in vitro activation-induced apoptosis of T lymphocytes in the patients with T1DM, depending on the state of compensation and duration of the disease. The features of activationinduced apoptosis have been studied in cultures of peripheral blood mononuclear cells (PBMC) in the patients with T1DM. Phytohemagglutinin (PHA) and insulin were used as apoptosis inducers. Increased in vitro sensitivity of PBMC to activation-induced apoptosis was revealed in T1DM patients. The strongest apoptotic response to PHA was detected in cases of T1DM decompensation. Considering predominantly Tcells to undergo apoptosis in response to PHA stimulation, one may speak about high sensitivity of activated T lymphocytes to induced apoptosis in the patients with T1DM. The highest level of activation-induced apoptosis in response to insulin stimulation was revealed in the compensation phase of T1DM. We have found that the intensity of spontaneous and activation-induced apoptosis correlates with decompensation of the disease and the degree of β-cells secretory function disorder. In fact, strong direct correlation was observed between the percentage of hypodiploid cells and blood concentration of glucose, and the inverse correlation was shown between the number of apoptotic cells and serum levels of C-peptide. The data obtained are in accordance with the modern concept of T1DM immunopathogenesis, which includes a development of autoimmune diseases associated not only with enhanced apoptosis of target cells, but also with a defect in phagocytic clearance of apoptotic cells due to impaired efferocytosis, i.e., phagocytosis of apoptotic cells. Thus, the maximal increase in spontaneous and activation-induced apoptosis levels of peripheral blood lymphocytes during the DM-1 decompensation is explained not only by the hyperglycemia effects, but also by the secondary immune response to the so-called “late apoptotic” or “secondary necrotic” β-cells, due to their ineffective phagocytic clearance.

ANTIAPOPTOSIS ACTIVITY OF PLANT POLYPRENYLPHOSPHATE AGAINST MACROPHAGE TARGET CELLS INFECTED WITH THE MURINE ENCEPHALOMYELITIS VIRUS

Antiapoptosis activity of plant polyprenylphosphate against macrophage target cells infected with the murine encephalomyelitis virus. Infection caused by the Theiler’s murine encephalomyelitis virus (TMEV) is regarded as an experimental model of multiple sclerosis, since both of these diseases are characterized by similar pathology of the central nervous system tissues and involvement of the immune system in the development of the demielinization. The aim of the work was to study the effect of plant-derived polyprenylphosphate (PP) on the apoptosis of infected target cells. We showed that PP reduced apoptosis of macrophage target cells infected with TMEV. It is known that in the protocol of multiple sclerosis treatment some medicines possessing immunomodulatory, antiviral, anti-inflammatory and antioxidant activity are used. Since PPs of plant origin also have all these activities, the prospects of their use as therapeutic agents are discussed.

Ultrasound-Sensitive Liposomes for Triggered Macromolecular Drug Delivery: Formulation and In Vitro Characterization.

Mistletoe lectin-1 (ML1) is a nature-derived macromolecular cytotoxin that potently induces apoptosis in target cells. Non-specific cytotoxicity to normal cells is one of the major risks in its clinical application, and we therefore propose to encapsulate ML1 in a nanocarrier that can specifically release its cargo intratumorally, thus improving the efficacy to toxicity ratio of the cytotoxin. We investigated the encapsulation of ML1 in ultrasound-sensitive liposomes (USL) and studied its release by high-intensity focused ultrasound (HAccessedIFU). USL were prepared by entrapment of perfluorocarbon nanodroplets in pegylated liposomes. The liposomes were prepared with different DPPC/cholesterol/DSPE-PEG2000 lipid molar ratios (60/20/20 for USL20; 60/30/10 for USL10; 65/30/5 for USL5) before combination with perfluorocarbon (PFC) nanoemulsions (composed of DPPC and perfluoropentane). When triggered with HIFU (peak negative pressure, 2–24 MPa; frequency, 1.3 MHz), PFC nanodroplets can undergo phase transition from liquid to gas thus rupturing the lipid bilayer of usl. Small unilamellar liposomes were obtained with appropriate polydispersity and stability. ML1 and the model protein horseradish peroxidase (HRP) were co-encapsulated with the PFC nanodroplets in USL, with 3% and 7% encapsulation efficiency for USL20 and USL10/USL5, respectively. Acoustic characterization experiments indicated that release is induced by cavitation. HIFU-triggered release of HRP from USL was investigated for optimization of liposomal composition and resulted in 80% triggered release for USL with USL10 (60/30/10) lipid composition. ML1 release from the final USL10 composition was also 80%. Given its high stability, suitable release, and ultrasound sensitivity, USL10 encapsulating ML1 was further used to study released ML1 bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging demonstrated its functional activity regarding the interaction with the target cells. We furthermore demonstrated the cytotoxicity of the released ML1 (I.E., After USL were treated with HIFU). The potent cytotoxicity (IC50 400 ng/ml; free ML1 IC50 345 ng/ml) was compared to non-triggered USL loaded with ML1. Our study shows that USL in combination with HIFU hold promise as trigger-sensitive nanomedicines for local delivery of macromolecular cytotoxins.

Downregulation of activin-signaling gene expression in passaged normal human dermal fibroblasts.

Activins are members of the transforming growth factor-β (TGF-β) superfamily and play important roles in proliferation, differentiation, and apoptosis of various target cells. We investigated changes of activin, activin receptor (ActR), and Smad-signaling gene expression with increasing passage number in normal human dermal fibroblasts. The expression of mRNA and protein was measured by reverse transcription-quantitative polymerase chain reaction and immunoblot analysis from passage numbers 5 to 15. Activin A and follistatin transcript levels increased with increasing passage number. ActR types IA, IB, IIA and IIB mRNA levels decreased at high passage number. The levels of Smad2, 3 and 4 protein decreased with increasing passage number, which also attenuated phosphorylation of Smad2 and 3 protein expression. Smad7 was enhanced with increasing passage number. These results suggest that expression of activin-signaling in aging normal human dermal fibroblasts increases activin A and follistatin, whereas ActR-Smad signaling is decreased.

Latent Membrane Protein 1 Contributes to Deficient Cellular Immunity in NKTCL Patients

Introduction Extranodal NK/T cell lymphoma (NKTCL) is an aggressive malignance that is correlated closely with persistent Epstein-Barr virus (EBV) infection and belongs to a latency II EBV disease. Chronic persistent viral infection is known to result in T cell exhaustion, which will dramatically impede anti-tumor immunity. Delineating mechanisms of immunosuppression contributes to the development of novel immuno-therapeutically strategies. Herein, we described the immune status and possible role of latent membrane protein 1(LMP1) in NKTCL. Methods Peripheral blood mononuclear cells (PBMCs) from newly diagnosed NKTCL patients and age-matched healthy donors (HDs) were isolated and stained with surface marker or intracellular marker after permeabilization. The frequency of EBV-specific cytotoxic T cells (CTLs) was analyzed by staining with HLA-A2 tetramers assembled with synthetic peptides from LMP1. T cell proliferation was detected by dilution of stained CFSE, and apoptosis of T cells was performed via annexinV and 7-AAD dual-staining. Cytotoxicity assay of natural killer cells (NK) was measured by detecting apoptosis of CFSE-labeled K562 cells after co-culturing with PBMCs. All flow cytometry was performed by BD FACS CantoII, and analyzed with FlowJo software. Results PBMCs from 19 NKTCL patients and 18 HDs were analyzed, which showed that the patients had higher ratio of CD4+/CD8+ cells and lower frequency of CD3-CD56+NK cells, higher percentage of immunosuppressive CD4+CD25hiCD127low T regulatory cells (Tregs) and HLA-DR-CD11b+CD33+ myeloid derived suppressor cells (MDSCs). Notably, the ratio of CD8+/Tregs, a parameter representing immune effector cells status, was obviously decreased in NKTCL patients (Figure-a). Given the potential association of T cell exhaustion and NKTCL, we detected expression of T cell exhaustion markers on T cells from NKTCL patients, and found that both CD4+ and CD8+ T cells expressed much higher level of inhibitory molecules PD1, CTLA4, TIGIT and TIM3 in NKTCL patients than that of HDs (Figure-b). To further explore cellular immunity in NKTCL, T cells were divided into four subsets: CD45RA-CCR7+ central memory T cells (Tcm), CD45RA+CCR7+ terminally differentiated effector T cells (Temra), CD45RA+CCR7- naïve T cells (Tn), CD45RA-CCR7- effector memory T cells (Tem) (Figure-c). It showed that CD4+ T cells had lower Tcm, Temra and Tn but much higher proportion of Tem compared with HDs (Figure-d). Moreover, we found that expression of PD1 and CTLA4 were upregulated on all lymphocyte subsets in NKTCL patients than HDs (Figure-e). We collected additional 13 NKTCL patients and 10 HDs with positive HLA-A2 phenotype to measure percentages of EBV-specific CTLs, which showed that frequency of EBV-specific CTLs was remarkably decreased in NKTCL patients (Figure-f). In addition, EBV-specific CTLs from NKTCL patients were more likely to express higher levels of exhaustion markers but produced much less IFN-γ (Figure g-h). To explore the potent mechanism of immunosuppression in NKTCL, LMP1 attracted our attention, which had been proven capable of activating multiple signaling pathways to exert its oncogenesis function. To elucidate effect of LMP1 on immune cells, we constructed two LMP1-derived peptides and found that LMP1-derived peptides suppressed the proliferation of CD4+ and CD8+ T cells and inhibited IFN-γ production of CD8+ T cells and NK cells (Figure i-j). Meanwhile, LMP1 promoted apoptosis of both CD4+ and CD8+ T cells (Figure-k). Subsequently regulation of LMP1 on exhaustion markers of T cells were detected, which showed that PD1, CTLA4, TIGIT and TIM3 were significantly upregulated on both CD4+ and CD8+ T cells after treatment with LMP1 derived peptides (Figure-m). Furthermore, LMP1 impaired NK cytotoxicity ability, evidenced by decrease apoptosis of target cells (Figure-n). Besides its effect on immune effector cells, we also found that LMP1-expressing NKTCL cell lines obviously induced both Tregs and MDSCs after co-culture. Interestingly, the extent of induction by LMP1 of Tregs and MDSCs were in line with the expression level of LMP1 on NKTCL cells (Figure o-p). Conclusion Our study showed that LMP1 contributes to deficient cellular immunity in NKTCL patients, providing us with more insight into immunosuppressive in this entity of disease, which would lead to identification of novel treatment strategies. Figure Disclosures No relevant conflicts of interest to declare.

The Anti-Leukemia Effect of Natural Killer-Derived Exosomes

Circulating NK cells are the effector arm of the innate immune system. As such, they recognize transformed cells as "non-self" and kill them. Numerous ex vivo studies have demonstrated the ability of NK cells to kill allogeneic leukemia cells. Based on these in vitro studies, several past and ongoing clinical trials have shown allogenic NK cells have a strong anti-leukemia effect. However, the use of NK cells as an "off the shelf" product is limited since they, like most cellular products, induce an allo-reactive immunogenic response which limits efficacy and increases toxicity. Exosomes are nano scaled extracellular vesicles that are released by various types of cells including NK cells. Since the exosomal cargo reflects, in part, the molecular makeup of its cell of origin, we hypothesized that NK-derived exosomes maintain the anti-leukemia effect of their cell of origin. To test this hypothesis, we exposed leukemia cells to NK-derived exosomes and tested their ability to eliminate them. As a source for NK-derived exosomes we used the NK-92MI cell line. This is a genetically altered cell line which constitutively expresses IL-2 that augments the cytotoxic activity of the cells and is routinely used in clinical trials. We cultured these cells in exosome free medium for 48 hours and extracted the exosomes by ultracentrifugation. Nanoparticle analyzing system showed that the pellet was enriched with the typical ~100nm sized vesicles and these particles were visualized by electron microscopy. Western immunoblotting confirmed that these particles express CD63, a known exosomal biomarker. Subsequently, we subjected CML K562, ALL Jurkat and AML HL-60 to NK-92MI-Exo stained with PKH-26 and showed by flow cytometry that these cells uptake the exosomes in a dose- and time- dependent manner. As controls, we used exosomes derived from the human embryonic kidney 293 (HEK-293) cell line. LDH release assay showed a marked increase in LDH activity in NK-92MI-Exo exposed leukemia cells but not in HEK-293-Exo exposed leukemia cells across all cell lines tested. Similarly, flow cytometry of leukemia cells double stained for annexin/PI showed that the rate of apoptosis was markedly increased in NK-92MI-Exo exposed leukemia cells but not in HEK-293-Exo exposed leukemia cells. Together, these assays indicate that NK-92MI-Exo are cytotoxic to various leukemia cell lines and that this effect is specific to NK-derived exosomes. Encouraged by our preliminary results, we harvested leukemia cells from 4 patients with acute myeloid leukemia (AML), 4 patients with acute lymphoblastic leukemia (ALL), 4 patients with chronic lymphocytic leukemia (CLL) and normal B-cells from healthy volunteers and exposed them to NK-92MI-Exo. Similar to the cell line results, we found a marked cytotoxic effect to NK-92MI-Exo on these leukemia cells but not on normal B-cells from healthy individuals whereas HEK-293-Exo had only a minimal or no effect on primary leukemia cells. Clonal assay on peripheral blood sample from 2 CML patients showed that the number of colony forming unit -granulocyte, erythrocyte, megakaryocyte monocyte (CFU-GEMM) is significantly reduced following exposure to NK-92MI-Exo, suggesting that these exosomes target leukemia progenitor cells. Proteomics analysis of NK-92MI-Exo revealed that similar to NK cytotoxic granules, the exosomal cargo include granozymes known to induce apoptosis of target cells but unlike NK cytotoxic granules the exosomal cargo does not include perphorines, known to perforate target cell membrane. Taken together our data suggests that NK-derived exosomes have a potent cytotoxic effect across a wide range of leukemia cells. This effect is specific to NK-exosomes. Furthermore, NK-derived exosomes preferentially kill leukemia cells but not normal cells. We also show that these exosomes are toxic to leukemia progenitor cells. Proteomics analysis suggested that NK-cytotoxic granules and exosomes use different strategies to enter target-cells. Whether NK-derived exosomes may become a-cellular therapeutic strategy to combat leukemia remains to be determined. Figure Disclosures No relevant conflicts of interest to declare.

Exploiting an Anti-CD3/CD33 Bispecific Antibody to Redirect Donor T Cells Against HLA Loss Leukemia Relapses

Background Genomic loss of mismatched HLAs ("HLA loss") represents a frequent modality by which acute myeloid leukemia (AML) evades immune recognition from donor T cells after partially HLA-incompatible allogeneic hematopoietic cell transplantation (allo-HCT). One important consequence of this post-transplantation relapse mechanism is that infusions of lymphocytes from the original donor become ineffectual, prompting the search for alternative therapeutic options. Here, to circumvent the loss of physiological T cell receptor-HLA interactions in these patients, we tested the ability of an anti-CD3/CD33 bispecific antibody (BsAb) to re-target donor T cells towards HLA loss relapses. Methods For short-term in vitro experiments, T cells were co-cultured with the MOLM-13 AML cell line or with primary patient blasts for 96 hours in presence or absence of an anti-CD3/CD33 BsAb. As readouts, we measured T cell activation (as surface expression of CD25 and CD69) and the absolute counts and relative proportion of effectors and targets. For long-term in vitro experiments, we established mixed lymphocyte cultures (MLCs) of T cells purified from two patients after haploidentical HCT and primary AML blasts obtained from the same patients at the time of diagnosis. After sequential stimulations, the co-cultures were tested against targets of interest, with or without addition of the BsAb. Functional readouts were T cell degranulation (measured as CD107a expression), antigen-specific activation (as CD137/41-BB expression) and target-specific cytotoxicity (measured by time-lapse live cell imaging over a 48 hour time span). For in vivo experiments, human leukemic cells were infused intravenously into non-irradiated NSG mice, followed by intraperitoneal infusion of T cells and daily administration of the BiTE compound. Results First, we retrospectively analyzed immunophenotypic data of 36 AML patients who experienced HLA loss relapses at our Institution, documenting robust expression of CD33 on the surface of the relapsed leukemia in 35 of them (97%; Figure 1A). By short-term co-culture experiments we titrated the BsAb concentration to be used for subsequent in vitro assays to 100 ng/ml, and the most informative effector:target ratio to 1:3. Then, we established MLCs by stimulating T cells collected from two patients after partially HLA-incompatible allo-HCT with AML blasts collected from the same patients at the time of diagnosis. In both cases, donor-derived T cells robustly responded against the patient blasts both in term of degranulation (Figure 1B) and of antigen-specific activation (Figure 1C). As expected, when we tested the same T cells against the patient leukemia at time of HLA loss relapse, we detected no T cell-mediated responses. Noticeably, when the BsAb was added, in both cases we detected a strong response not only against the diagnosis but also against the HLA loss variants, indicating that T cells were effectively re-targeted towards leukemic cells. Similar results were obtained also by live cell imaging, measuring target cell apoptosis over 48 hours of recording: also in this assay, in fact, donor T cells recognized and killed leukemia at diagnosis (45% of detection area positive for apoptosis dye) and failed to recognize its HLA loss relapse counterpart (32% of area positive for apoptosis dye). Addition of the BsAb to the co-cultures had a minor effect on recognition of the original disease (45% of area positive for apoptosis dye) but drove dramatic cell death of HLA loss blasts (80% of area positive for apoptosis dye), demonstrating that the BsAb induced not only T cell activation but also and most importantly target cell killing (Figure 1D). Finally, we modeled the BsAb activity in vivo, showing that, whereas the sole infusion of human T cells is not able to prevent the outgrowth of leukemia in the bone marrow of NSG mice, addition of the bispecific antibody leads to effective disease clearance (Figure 1E). Conclusions Our results demonstrate that anti-CD3/CD33 BsAbs can effectively redirect donor T cells against HLA loss leukemia variants, resulting in their rapid and effective killing. Taken together, these promising findings strongly support translation of this approach to ad hoc designed early-phase clinical trials, to provide a rational therapy for this increasingly recognized but still treatment-orphan modality of post-transplantation relapse. Figure 1 Disclosures Subklewe: Janssen: Consultancy; Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Oxford Biotherapeutics: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Roche: Consultancy, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding. Vago:Moderna Therapeutics: Research Funding; GenDx: Research Funding.

Soluble FAS ligand is not required for pancreatic islet inflammation or beta-cell destruction in non-obese diabetic mice

CD8+ T cells play a central role in beta-cell destruction in type 1 diabetes. CD8+ T cells use two main effector pathways to kill target cells, perforin plus granzymes and FAS ligand (FASL). We and others have established that in non-obese diabetic (NOD) mice, perforin is the dominant effector molecule by which autoreactive CD8+ T cells kill beta cells. However, blocking FASL pharmacologically was shown to protect NOD mice from diabetes, indicating that FASL may have some role. FASL can engage with its receptor FAS on target cells either as membrane bound or soluble FASL. It has been shown that membrane-bound FASL is required to stimulate FAS-induced apoptosis in target cells, whereas excessive soluble FASL can induce NF-κB-dependent gene expression and inflammation. Because islet inflammation is a feature of autoimmune diabetes, we tested whether soluble FASL could be important in disease pathogenesis independent of its cell death function. We generated NOD mice deficient in soluble FASL, while maintaining expression of membrane-bound FASL due to a mutation in the FASL sequence required for cleavage by metalloproteinase. NOD mice lacking soluble FASL had normal numbers of lymphocytes in their spleen and thymus. Soluble FASL deficient NOD mice had similar islet inflammation as wild-type NOD mice and were not protected from diabetes. Our data indicate that soluble FASL is not required in development of autoimmune diabetes.

Combination Suicide Gene Delivery with an Adeno-Associated Virus Vector Encoding Inducible Caspase-9 and a Chemical Inducer of Dimerization Is Effective in a Xenotransplantation Model of Hepatocellular Carcinoma.

Current treatment approaches for hepatocellular carcinoma (HCC) have a narrow therapeutic index and alternate modes of treatment are thus required. We have utilized a gene delivery vector containing inducible caspase 9 (iCasp9) gene, which is a synthetic analogue based on the mammalian caspase 9 and fused to a human FK506 binding protein that allows its conditional dimerization to a synthetic, small molecule [chemical inducer of dimerization, AP20187] and results in target cell apoptosis. In our studies, we have tested these synthetic vectors based on an adeno-associated virus platform for their potential anti-tumorigenic effect in human HCC cells in vitro and in a HCC tumor model developed in nude mice. Our data demonstrates that the iCasp9-AP20187 bioconjugate is able to trigger terminal effectors of cellular apoptosis and presents a viable approach for the potential treatment of HCC.

Anti-inflammatory activity of extensively hydrolyzed casein is mediated by granzyme B.

Nutritional factors such as extensively hydrolyzed casein (eHC) have been proposed to exert anti-inflammatory activity and affect clinical outcomes such as tolerance development in cow's milk allergy. Granzyme B (GrB) induces apoptosis in target cells and also controls the inflammatory response. Whether eHC could affect the activity of granzyme B and play a role in GrB-mediated inflammatory responses in vitro was unknown. The activity of GrB was measured using the substrate Ac-IEPD-pNA. Inflammatory responses were induced with GrB in HCT-8 and THP-1 cells, and pro-inflammatory cytokines were determined at the transcriptional and protein level. GrB could induce the expression of IL-1β in HCT-8 cells, and IL-8 and MCP-1 in THP-1 cells, respectively. Interestingly, GrB acted synergistically on LPS-induced inflammation in HCT-8 cells and eHC reduced pro-inflammatory responses in both GrB and LPS-mediated inflammation. Further analyses revealed that eHC could inhibit the biological activities and cytotoxic activities of GrB and then could reduce GrB-mediated inflammatory response. The results from the current study suggest that anti-inflammatory activity of extensively hydrolyzed casein is, to a certain extent, mediated through modulation of granzyme B activity and responses.

Automated Classification of Apoptosis in Phase Contrast Microscopy Using Capsule Network.

Automatic and accurate classification of apoptosis, or programmed cell death, will facilitate cell biology research. The state-of-the-art approaches in apoptosis classification use deep convolutional neural networks (CNNs). However, these networks are not efficient in encoding the part-whole relationships, thus requiring a large number of training samples to achieve robust generalization. This paper proposes an efficient variant of capsule networks (CapsNets) as an alternative to CNNs. Extensive experimental results demonstrate that the proposed CapsNets achieve competitive performances in target cell apoptosis classification, while significantly outperforming CNNs when the number of training samples is small. To utilize temporal information within microscopy videos, we propose a recurrent CapsNet constructed by stacking a CapsNet and a bi-directional long short-term recurrent structure. Our experiments show that when considering temporal constraints, the recurrent CapsNet achieves 93.8% accuracy and makes significantly more consistent prediction than NNs.

Intrinsic factor Recognition Promotes Th17/Th1 Autoimmune Gastric Inflammation in Patients with Pernicious Anemia

Abstract The intrinsic factor (IF) is the major humoral autoantigen in pernicious anemia (PA)/autoimmune gastritis. Although many studies have examined the autoantibody response to intrinsic factor and H+,K+-ATPase, no information is available on possible pathogenic mechanisms mediated by IF-specific gastric T cells. The aim of this study was to investigate IF-specific T cells in the gastric mucosa of PA patients and define their functional properties. In vivo-activated T cells from the infiltrates of the gastric mucosa of PA patients were isolated and cloned. We investigated the ability of the gastric T-cell clones to proliferate and produce cytokines in response to IF, as well as their helper function for antibody production and their cytolytic potential. For the first time we provide evidence that gastric mucosa of PA patients harbour a high proportion (20%) of autoreactive activated CD4+ T-cell clones that specifically recognize IF. Most of these clones (94%) showed a Th17 or Th1 profile. Virtually all IF-specific clones produced TNFα, IL-21 and provided substantial help for B-cell immunoglobulin production. Most mucosa-derived IF-specific T-cell clones expressed perforin-mediated cytotoxicity and induced Fas-Fas ligand-mediated apoptosis in target cells. Altogether, our results suggest that chronic autoantigen-induced T cell– dependent B-cell activation is responsible for the local synthesis of IF autoantibodies found in the sera and indicate that activation of IF-specific Th17 and Th1 T cells in the gastric mucosa represent a key effector mechanism in PA suggesting that the Th17/Th1 pathway may represent a novel target for the prevention and treatment of the disease.

Extracellular vesicles derived from natural killer cells use multiple cytotoxic proteins and killing mechanisms to target cancer cells

ABSTRACTExtracellular vesicles (EVs) are secreted membrane vesicles, which play complex physiological and pathological functions in intercellular communication. Recently, we isolated natural killer (NK) cell-derived EVs (NK-EVs) from ex vivo expansion of NK cell cultures. The isolated NK-EVs contained cytotoxic proteins and several activated caspases, and they induced apoptosis in target cells. In this report, the protein levels of cytotoxic proteins from NK-EV isolates were analysed by ELISA. The mean values of perforin (PFN, 550 ng/mL), granzyme A (GzmA, 185 ng/mL), granzyme B (GzmB, 23.4 ng/mL), granulysin (GNLY, 56 ng/mL), and FasL (2.5 ng/mL) were obtained from >60 isolations using dot plots. The correlation between cytotoxicity and cytotoxic protein levels was examined by linear regression. PFN, GzmA, GzmB, GNLY all had a positive, moderate correlation with cytotoxicity, suggesting that there is not a single cytotoxic protein dominantly involved in killing and that all of these proteins may contribute to cytotoxicity. To further explore the possible killing mechanisms, cells were treated with NK-EVs, proteins extracted and lysates assessed by Western blotting. The levels of Gzm A substrates, SET and HMG2, were diminished in targeted cells, indicating that GzmA may induce a caspase-independent death pathway. Also, cytochrome C was released from mitochondria, a central hallmark of caspase-dependent death pathways. In addition, several ER-associated proteins were altered, suggesting that NK-EVs may induce ER stress resulting in cell death. Our results indicate that multiple killing mechanisms are activated by NK-derived EVs, including caspase-independent and -dependent cell death pathways, which can mediate cytotoxicity against cancer cells.Abbreviations: NK: natural killer cells; aNK: activated NK cells; EV: extracellular vesicles; ER: endoplasmic reticulum; ALL: acute lymphoblastic leukaemia; FBS: foetal bovine serum. GzmA: granzyme A; GzmB: granzyme B; GNLY: granulysin; PFN: perforin

Dynamic Synthetic Biointerfaces: From Reversible Chemical Interactions to Tunable Biological Effects.

Dynamic synthetic biointerface is a new concept of biomaterials with smart surface properties capable of controlled display of bioactive ligands, dynamic modulation of cell-biomaterial interactions, and subsequently clever manipulation of fundamental cell behaviors like adhesion, migration, proliferation, differentiation, apoptosis, and so on. As mimics of the extracellular matrix (ECM), such molecularly dynamic biointerfaces have attracted increasing attention because of their tunable biological effects with great significance in in situ cell biology, tissue engineering, drug targeting, and cell isolation for cancer theranostics. Approaches to control bioligand presentation on materials mainly rely on surface functionalization with dynamic or reversible chemical linkers to which the ligands are tethered. Photoelectric-transformable or photocleavable chemistry, host-guest supramolecular chemistry, and multiple noncovalent interactions were initially employed for fabrication of dynamic synthetic biointerfaces. However, the external stimuli required in these systems, including electrochemical potential, electrochemical reaction, and near-infrared or UV light, are mostly invasive to living cells; and few of them are able to respond to the stimuli occurring in natural biological processes. In addition, most of current systems focused only on the control of cell adhesion, other cell behaviors like migration, differentiation and apoptosis have rarely been explored. Therefore, the development of novel synthetic biointerfaces that permit access to noninvasive control of diverse cell behaviors still represents a key challenge in biomaterials science. Our group pioneers the use of reversible covalent bonds, metal coordinative interactions, and the molecular affinity of molecularly imprinted synthetic receptors as the dynamic driving forces for the fabrication of smart biointerfaces. Several typical biological stimuli, such as glycemic volatility, body temperature fluctuations, regional disparity of pH values, and specific biomolecules, were tactfully involved in our systems. In this Account, we highlight the strategies we have used on the exploitation of dynamic synthetic biointerfaces based on the above three types of reversible chemical interactions. While our attention has been focused on biologically stimuli-responsive or other noninvasive ligand presentation, the versatility of dynamic synthetic biointerfaces in control of cell adhesion, directing cell differentiation, and targeting cell apoptosis has also been successfully demonstrated. In addition, a paradigm shift of dynamic synthetic biointerfaces from macroscopic to microscopic scale (e.g., nanobiointerfaces) was conceptually demonstrated in our research. The potential applications of these developed dynamic systems, including fundamental cell biology, surface engineering of biomaterials, scaffold-free tissue engineering, cell-based cancer diagnosis, and drug targeting cancer therapy, were also introduced, respectively. Although the development of dynamic synthetic biointerfaces is still in its infancy, we strongly believe that further efforts in this field will play a continuously and increasingly significant role in bridging the gap between chemistry and biology.

Orthohantaviruses belonging to three phylogroups all inhibit apoptosis in infected target cells

Orthohantaviruses, previously known as hantaviruses, are zoonotic viruses that can cause hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) in humans. The HPS-causing Andes virus (ANDV) and the HFRS-causing Hantaan virus (HTNV) have anti-apoptotic effects. To investigate if this represents a general feature of orthohantaviruses, we analysed the capacity of six different orthohantaviruses – belonging to three distinct phylogroups and representing both pathogenic and non-pathogenic viruses – to inhibit apoptosis in infected cells. Primary human endothelial cells were infected with ANDV, HTNV, the HFRS-causing Puumala virus (PUUV) and Seoul virus, as well as the putative non-pathogenic Prospect Hill virus and Tula virus. Infected cells were then exposed to the apoptosis-inducing chemical staurosporine or to activated human NK cells exhibiting a high cytotoxic potential. Strikingly, all orthohantaviruses inhibited apoptosis in both settings. Moreover, we show that the nucleocapsid (N) protein from all examined orthohantaviruses are potential targets for caspase-3 and granzyme B. Recombinant N protein from ANDV, PUUV and the HFRS-causing Dobrava virus strongly inhibited granzyme B activity and also, to certain extent, caspase-3 activity. Taken together, this study demonstrates that six different orthohantaviruses inhibit apoptosis, suggesting this to be a general feature of orthohantaviruses likely serving as a mechanism of viral immune evasion.

Kaempferol Improves TRAIL-Mediated Apoptosis in Leukemia MOLT-4 Cells by the Inhibition of Anti-apoptotic Proteins and Promotion of Death Receptors Expression.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a member of the Tumor Necrosis Factor (TNF) superfamily, which stimulates apoptosis in a wide range of cancer cells through binding to Death Receptors 4 and 5 (DR4/5). Nevertheless, TRAIL has noticeable anti-cancer abilities; some cancer cells acquire resistance to TRAIL, and consequently, its potential for inducing apoptosis in target cells is strongly diminished. Acute lymphoblastic leukemia MOLT-4 cell line is one of the most resistant cells to TRAIL that developed resistance to TRAIL through different pathways. TRAIL plus kaempferol was used to eliminate the resistance of the MOLT-4 cells to TRAIL. Firstly, IC50 for kaempferol (95μM) was determined by using the MTT assay. Secondly, the viability of the MOLT-4 cells was assayed by FACS after Annexin V/PI staining, following treatment with TRAIL (50 and 100nM) and kaempferol (95μM) alone and in combination. Finally, the expression levels of the candidate genes involved in resistance to TRAIL were assayed by real-time PCR technique. Kaempferol plus TRAIL induced apoptosis robustly in MOLT-4 cells at 12, 24 and 48 hours after treatment. Additionally, it was found that kaempferol could inhibit the expression of c-FLIP, X-IAP, cIAP1/2, FGF-8 and VEGF-beta, and conversely augment the expression of DR4/5 in MOLT-4 cells. It is suggested that co-treatment of MOLT-4 cells with TRAIL plus kaempferol is a practical and attractive approach to eliminate cancers' resistance to TRAIL by inhibition of the intracellular anti-apoptotic proteins, upregulation of DR4/5 and also by suppression of the VEGF-beta (VEGFB) and FGF-8 expressions.

Venetoclax Enhances the Efficacy of Therapeutic Antibodies in B-Cell Malignancies

The application of antibodies is a promising option in the treatment of B-cell malignancies, including acute lymphoblastic leukemia (ALL) and B-cell Non-Hodgkin lymphoma (B-NHL). Although patient outcomes have improved by applying combinations of chemotherapy and antibodies, certain patients characterized by a high expression of anti-apoptotic Bcl-2 have a poor prognosis. These include adult B-NHL patients with “double-hit lymphomas” (DHLs) and pediatric ALL patients harboring a t(17;19) translocation. Furthermore, a substantial number of Burkitt´s lymphoma (BL) patients also express Bcl-2 even though the impact of this finding on prognosis is yet unclear.Here, we examine the role of low doses of the Bcl-2 inhibitor venetoclax (VTX, 1nM) on the efficacy of the therapeutic antibodies rituximab (CD20), daratumumab (CD38) and CD19-DE (a variant of the CD19 antibody MOR208 engineered for improved effector cell binding). Natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) were evaluated. The CD20-expressing DHL cell line Carnaval and patient-derived xenograft (PDX) cells from two BL patients were used as target cells for rituximab, the CD20-negative/CD38-positive DHL cell line Will-2 was used with daratumumab and three PDX samples from t(17;19) positive ALL patients were used with CD19-DE. For the assessment of ADCP, human monocyte-derived macrophages were incubated with labelled target cells and microscopy assays were performed.NK-cell mediated ADCC was not enhanced by VTX in any of our models. However, 17-37% increases in ADCP by macrophages were detected when Carnaval cells were subjected to combinations of VTX/rituximab and when Will-2 cells were treated with VTX/daratumumab as compared to VTX or antibody alone (p=0.0318/p=0.0185 and p=0.0012/p=0.0068, respectively, Figure A). When BL PDX cells were subjected to ADCP assays with VTX/rituximab, mean phagocytosis levels were also enhanced by 26.0% and 21.0% in the combination treatment group as compared to VTX (p=0.0283) and rituximab alone (p=0.0282; Figure A). ADCP assays with t(17;19) positive ALL-PDX cells and CD19-DE confirmed these results as phagocytosis was increased to similar extents in the combination group as compared to VTX (p=0.0017) or CD19-DE alone (p=0.0323) (Figure A/B). In order to exclude that our observations were due to an enhancement of apoptosis in target cells only, we measured cleaved caspase-3 with VTX, antibodies alone or the combination of both. Cleaved caspase-3 levels were equal in all groups suggesting that the addition of VTX resulted in an apoptosis-independent activation of macrophages. In order to minimize heterogeneity in ADCP assays, phagocytosis was next examined using expanded macrophages from NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice for our assays. Regardless of target cells and antibody, the combination of antibody treatment with VTX resulted in enhanced phagocytosis by murine macrophages, confirming our results.The effects of VTX on the efficacy of rituximab were finally examined in vivo. Carnaval cells were injected intravenously into NSG mice and animals treated with VTX (100 mg/kg 5 days/week by oral gavage), rituximab alone (1 mg/kg once weekly intraperitoneally) or the combination of both (n=6/group). Mice were sacrificed when mice showed clinical lymphoma or leukemia engraftment and survival differences were assessed using Kaplan-Meier log-rank statistics. Compared to control, mice treated with VTX displayed a slight survival advantage, which was more marked in mice treated with rituximab (p=0.0020/p=0.0004, respectively, Figure C), suggesting a better efficacy of rituximab than VTX as monotherapy. Most importantly, mice treated with the combination VTX/rituximab showed significantly superior survival as compared to either VTX or rituximab alone (p=0.0023/p=0.0268, respectively, Figure C), suggesting additive effects in vivo.Altogether, we show that VTX enhances the efficacy of therapeutic antibodies in models of B-cell malignancies including PDX samples. The mechanism is most likely dependent on distinct influences of VTX on macrophage activation, e. g. by myeloid immune checkpoints. Our in vivo data suggest that this combination strategy may become a promising therapeutic option for the treatment of Bcl-2 expressing B-cell malignancies in the future. [Display omitted] DisclosuresBourquin:Amgen: Other: Travel Support. Valerius:Affimed: Research Funding. Peipp:Affimed: Research Funding.