Cutting edge: contributions of apoptosis and anergy to systemic T cell tolerance

Multiple pathways can induce and maintain peripheral T cell tolerance. The goal of this study was to define the contributions of apoptosis and anergy to the maintenance of self-tolerance to a systemic Ag. Upon transfer into mice expressing OVA systemically, OVA-specific DO11 CD4+ T cells are activated transiently, cease responding, and die. Bim is the essential apoptosis-inducing trigger and apoptosis proceeds despite increased expression of Bcl-2 and Bcl-x. However, preventing apoptosis by eliminating Bim does not restore proliferation or cytokine production by DO11 cells. While Foxp3 is transiently induced, anergy is not associated with the stable development of regulatory T cells. Thus, apoptosis is dispensable for tolerance to a systemic self-Ag and cell-intrinsic anergy is sufficient to tolerize T cells.     

RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8+ T cells

Pancreatic cancer is characterized by a microenvironment suppressing immune responses. RIG-I-like helicases (RLH) are immunoreceptors for viral RNA that induce an antiviral response program via the production of type I interferons (IFN) and apoptosis in susceptible cells. We recently identified RLH as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumor cell death. Treatment of murine pancreatic cancer cell lines with RLH ligands induced production of type I IFN and proinflammatory cytokines. In addition, tumor cells died via intrinsic apoptosis and displayed features of immunogenic cell death, such as release of HMGB1 and translocation of calreticulin to the outer cell membrane. RLH-activated tumor cells led to activation of dendritic cells (DCs), which was mediated by tumor-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. Importantly, CD8α⁺ DCs effectively engulfed apoptotic tumor material and cross-presented tumor-associated antigen to naive CD8⁺ T cells. In comparison, tumor cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation and antigen presentation. Tumor cells treated with sublethal doses of RLH ligands upregulated Fas and MHC-I expression and were effectively sensitized towards Fas-mediated apoptosis and cytotoxic T lymphocyte (CTL)-mediated lysis. Vaccination of mice with RLH-activated tumor cells induced protective antitumor immunity in vivo. In addition, MDA5-based immunotherapy led to effective tumor control of established pancreatic tumors. In summary, RLH ligands induce a highly immunogenic form of tumor cell death linking innate and adaptive immunity.Patients diagnosed with pancreatic cancer face a poor prognosis due to early metastasis and therapy resistance, resulting in a 5-year survival rate of only 6%.¹ Treatment options for inoperable tumors are limited and offer little benefit for the patients. But even after tumor resection most patients relapse and succumb to their disease, as evidenced by a 5-year survival rate of 20%.² Novel treatment strategies such as immunotherapy are being investigated.³ Pancreatic cancer is characterized by an immunosuppressive microenvironment, which is mediated by cytokines such as TGF-β, modulation of antigen-presenting cells, impaired T-cell effector function as well as recruitment of regulatory T cells and myeloid-derived suppressor cells.⁴ Immunosuppressive factors correlate with a poor prognosis for patients with pancreatic cancer.^{5, 6, 7, 8} On the other hand, T-cell infiltrates of the tumor were found to be a positive prognostic factor.⁹ The major challenge for immunotherapy will be to counteract immunosuppressive mechanisms for tipping the balance toward productive immune responses against the tumor.Tumor cell death occurs spontaneously in fast growing tumors or is induced by specific therapies, such as cytotoxic agents or irradiation. Several forms of cell death, such as apoptosis, necrosis, autophagy, mitotic catastrophe and senescence can be discriminated. It appears that the conditions leading to tumor cell death dictate immunological consequences.^{10, 11} In most circumstances, cell death is immunologically silent, leading to tolerance rather than immunity. In specific situations, dying cells release immunogenic signals to the cell surface or the extracellular space leading to the activation of antigen-presenting cells, such as DCs, and facilitating antigen uptake and presentation. These signals are collectively called danger-associated molecular patterns (DAMPs) and include calreticulin exposure on the outer cell membrane, release of heat shock proteins, HMGB1, DNA, RNA, ATP and uric acid crystals, or the secretion of proinflammatory cytokines, such as IL-1 and IL-6.¹² Evidence has accumulated that certain chemotherapeutic drugs, which were traditionally considered to mediate antitumor effects via their antiproliferative properties, induce an immunogenic form of cell death leading to tumor-directed immunity.^{11, 13}Immune responses against viruses share many features with those against tumors. Mimicking a viral infection can be exploited for tumor immunotherapy. Double-stranded viral RNA is recognized by cytosolic pattern recognition receptors called RIG-I-like helicases (RLH), including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated antigen 5 (MDA5).^{14, 15, 16} Synthetic RLH ligands include 5′-triphosphate RNA (ppp-RNA) for RIG-I, and polyinosinic:polycytidylic acid (poly(I:C)) for MDA5. RLH initiate a signaling cascade mediated by IFN regulatory factor 3 (IRF-3), IRF-7 and NF-κB, leading to an antiviral response program characterized by the production of type I IFN and other innate immune response genes.^{17, 18} In addition, RLH signaling induces intrinsic apoptosis in tumor cells, which are highly susceptible, as compared with nonmalignant cells.^{19, 20} RLH ligands have been evaluated as therapeutic agents in preclinical tumor models for melanoma, ovarian cancer and pancreatic cancer.^{19, 21, 22, 23, 24} Therapeutic efficacy was enhanced by combining RNAi-mediated gene silencing with RIG-I activation in a single RNA molecule.^{21, 24} A ppp-siRNA targeting the anti-apoptotic protein Bcl-2 to promote tumor apoptosis showed therapeutic efficacy in experimental melanoma.²⁴ In this model, the antitumor effect was dependent on NK cells. To counteract tumor-induced immunosuppression, our group generated a ppp-siRNA silencing TGF-β1, which showed therapeutic efficacy in an orthotopic model of pancreatic cancer.²¹ Interestingly, with this approach CD8⁺ T cells mediated antitumor efficacy. Others reported that treatment of human ovarian cancer cells with RLH ligands resulted in phagocytosis of apoptotic tumor cells by monocyte-derived DCs and DC activation.^{22, 23} Together, these findings indicate that RLH-induced tumor cell death may promote adaptive immunity against tumors. However, mechanisms leading to DC activation and the impact on tumor antigen cross-presentation by DCs, which defines immunogenic cell death, have not been explored.In this study, we investigated the effects of RLH-induced tumor cell death on DC activation, antigen uptake and cross-presentation of tumor antigen by primary murine DC populations. We also studied mechanisms leading to DC activation using mice deficient in pathways of TLR, RAGE, inflammasome and type I IFN signaling. In addition, we assessed the immunogenicity and therapeutic efficacy of RLH-based immunotherapy in two different mouse models for pancreatic cancer.     

A lycopene β‐cyclase/lycopene ε‐cyclase/light‐harvesting complex‐fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α‐carotene and β‐carotene at different ratios

Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.     

Abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in a murine model of systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by oxidative stress, impaired vascular function, and attenuated angiogenesis. The tight-skin (Tsk(-/+)) mouse is a model of SSc that displays many of the cellular features of the clinical disease. We tested the hypotheses that abnormal fibrillin-1 expression and chronic phospholipid oxidation occur in Tsk(-/+) mice and, furthermore, that these factors precipitate a prooxidant state, collagen-related protein expression, apoptosis, and mesenchymal transition in endothelial cells cultured on Tsk(-/+) extracellular matrix. Human umbilical vein endothelial cells were seeded on microfibrils isolated from skin of C57BL/6J (control) and Tsk(-/+) mice in the presence or absence of chronic pretreatment with the apolipoprotein Apo A-I mimetic D-4F (1 mg·kg(-1)·day(-1) ip for 6 to 8 wk). Nitric oxide-to-superoxide anion ratio was assessed 12 h after culture, and cell proliferation, apoptosis, and phenotype were studied 72 h after culture. Tsk(-/+) mice demonstrated abnormal "big fibrillin" expression (405 kDa) by Western blot analysis compared with control. Endothelial cells cultured on microfibrils prepared from Tsk(-/+) mice demonstrated reduced proliferation, a prooxidant state (reduced nitric oxide-to-superoxide anion ratio), increased apoptosis, and collagen-related protein expression associated with mesenchymal transition. Chronic D-4F pretreatment of Tsk(-/+) mice attenuated many of these adverse effects. The findings demonstrate that abnormal fibrillin-1 expression and chronic oxidative stress mediate endothelial mesenchymal transition in Tsk(-/+) mice. This mesenchymal transition may contribute to the reduction in angiogenesis that is known to occur in this model of SSc.     

Colonic carcinogenesis along different genetic routes: glycophenotyping of tumor cases separated by microsatellite instability/stability

Different genetic routes account for colonic carcinogenesis. However, when analyzing colon cancer specimens, separation into different groups based on genetic alterations is commonly not performed. Thus, we here initiate the comparative phenotyping considering microsatellite instability/stability for clinical specimens. The focus is given to glycan epitopes, expression of which is known to be modulated by signal-transducing proteins that act as key regulators of normal colon epithelial growth and differentiation. In addition to six plant lectins used as sensors, the presence of two adhesion/growth-regulatory galectins is studied. Overall, a considerable level of intra- and interindividual heterogeneity is revealed. Alterations in the proportion of stained cells between tumor-adjacent and malignant epithelia concerned plant lectins, which bind substituted N-glycan cores, α2,6-sialylated branch ends, core 1 O-glycans and N-acetylgalactosamine. A tendency for changes was noted between microsatellite-unstable and microsatellite-stable cases for core substitution (bisected N-glycan, presence of β1,6-branching) and status of α2,6-sialylation. Statistical significance was reached for presence of galectin-3, found to be elevated in microsatellite-stable compared to microsatellite-unstable tumors. These results emphasize the potential of distinct signaling pathways to regulate certain aspects of the glycophenotype in vivo and thus delineate a perspective to discern functionally relevant deviations in expression of endogenous lectins and their counter-receptors.     

Muscle transcriptomic analyses in Angus cattle with divergent tenderness

Beef tenderness contributes significantly to variation of beef palatability, and is largely influenced by various genetic and environmental factors. To identify candidate genes and pathways related to beef tenderness, we analyzed the longissimus dorsi (LD) muscle of Angus cattle that had different degrees of tenderness, measured by Warner-Bratzler shear force (WBSF). Microarray and RT-PCR analyses identified 53 genes that were differentially expressed in LD samples categorized as either tough or tender, including myosin, heavy chain 3 skeletal muscle embryonic (MYH3), myosin heavy chain 8 skeletal muscle perinatal (MYH8), guanylate binding protein 5 (GBP5), fatty acid binding protein 4 (FABP4), Stearoyl-coenzyme A desaturase (SCD), Fatty acid synthase (FASN), ubiquitin-like with PHD and ring finger domains 1 (UHRF1). Most of these genes are involved in lipid metabolism and skeletal muscle contraction. Employing Gene ontology (GO) and Ingenuity Pathway Analysis (IPA), several GO terms and pathways were found to be related to hydrolase, peptidase and GTPase activity, lipid metabolism, small molecule biochemistry, molecular transport, and tissue development. Overall, this analysis provides insight into the metabolic relationships between muscle biology and beef quality.     

Monitoring neuronal calcium signalling using a new method for ratiometric confocal calcium imaging

Ca2+ signalling influences many processes in the adult and developing nervous system like exocytosis, synaptic plasticity, and growth cone motility. Optical techniques in combination with fluorescent Ca2+ indicators are the most frequently used methods to measure Ca2+ signalling in cells. In the present study, a new method for ratiometric confocal Ca2+ imaging was developed, and the usefulness of the system was tested with two different neuronal preparations. Developing Manduca sexta antennal lobe neurons were loaded with the Ca2+-sensitive dye Fura Red-AM, and the ratio of fluorescence excited at 457 and 488nm was measured with a confocal laser scanning microscope. During pupal stages 4-12, the antennal lobe neuropil is restructured which includes the ingrowth of olfactory receptor axons, dendritic outgrowth of antennal lobe neurons, and synaptogenesis. In antennal lobe neurons, application of the AChR agonist carbachol induced Ca2+ oscillations the amplitude and frequency of which changed during stages 4-9, while at the end of synaptogenesis, at stages 11 and 12, only single Ca2+ transients were elicited. The Ca2+ oscillations were blocked by D-tubocurarine and Cd2+, indicating that they were due to Ca2+ influx through voltage-gated Ca2+ channels, activated by nAChR-mediated membrane depolarization. To test whether single action potentials can induce Ca2+ transients detectable by Fura Red, individual leech Retzius neurons were injected iontophoretically with the Ca2+ indicator, and the membrane potential was recorded during Ca2+ imaging. Single action potentials induced transient increases in the Fura Red ratio measured in the axon, while trains of action potentials elicited Ca2+ transients that could also be recorded in the cell body and the nucleus. The results show that Fura Red can be used as a ratiometric Ca2+ indicator for confocal imaging.     

Nucleosomal arrays can be salt-reconstituted on a single-copy MMTV promoter DNA template: their properties differ in several ways from those of comparable 5S concatameric arrays

Subsaturated nucleosomal arrays were reconstituted on a single-copy MMTV promoter DNA fragment by salt dialysis procedures and studied by atomic force microscopy. Up to an occupation level of approximately eight nucleosomes on this 1900 bp template, salt reconstitution produces nucleosomal arrays which look very similar to comparably loaded 5S rDNA nucleosomal arrays; i.e., nucleosomes are dispersed on the DNA template. Thus, at these occupation levels, the single-copy MMTV template forms arrays suitable for biophysical analyses. A quantitative comparison of the population features of subsaturated MMTV and 5S arrays detects differences between the two: a requirement for higher histone levels to achieve a given level of nucleosome occupation on MMTV templates, indicating that nucleosome loading is thermodynamically less favorable on this template; a preference for pairwise nucleosome occupation of the MMTV (but not the 5S) template at midrange occupation levels; and an enhanced salt stability for nucleosomes on MMTV versus 5S arrays, particularly in the midrange of array occupation. When average occupation levels exceed approximately eight nucleosomes per template, MMTV arrays show a significant level of mainly intramolecular compaction; 5S arrays do not. Taken together, these results show clearly that the nature of the underlying DNA template can affect the physical properties of nucleosomal arrays. DNA sequence-directed differences in the physical properties of chromatin may have important consequences for functional processes such as gene regulation.