[Show abstract][Hide abstract] ABSTRACT: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.
For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.
Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.
[Show abstract][Hide abstract] ABSTRACT: Immune responses against cancer cells are often hindered by immunosuppressive mechanisms that are developed in the tumor microenvironment. Induction of a hyporesponsive state in tumor Ag-specific T cells is one of the major events responsible for the inability of the adaptive immune system to mount an efficient antitumor response and frequently contributes to lessen the efficacy of immunotherapeutic approaches. Treatment of localized tumors by focused ultrasound (FUS) is a minimally invasive therapy that uses a range of input energy for in situ tumor ablation through the generation of thermal and cavitation effect. Using a murine B16 melanoma tumor model, we show that a variant of FUS that delivers a reduced level of energy at the focal point and generates mild mechanical and thermal stress in target cells has the ability to increase immunogenic presentation of tumor Ags, which results in reversal of tumor-induced T cell tolerance. Furthermore, we show that the combination of nonablative low-energy FUS with an ablative hypofractionated radiation therapy results in synergistic control of primary tumors and leads to a dramatic reduction in spontaneous pulmonary metastases while prolonging recurrence-free survival only in immunocompetent mice.
Full-text · Article · Jan 2016 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative l-asparaginase exhibits l-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that l-asparagine is a nutrient important for T cell activation and that l-asparagine deprivation, such as that mediated by the Salmonella Typhimurium l-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and l-lactate secretion. We also show that l-asparagine deprivation mediated by the Salmonella Typhimurium l-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming.
No preview · Article · Oct 2015 · Journal of leukocyte biology
[Show abstract][Hide abstract] ABSTRACT: Macroautophagy is a cellular process that mediates the degradation in the lysosome of cytoplasmic components including proteins and organelles. Previous studies have shown that macroautophagy is induced in activated T cells to regulate organelle homeostasis and the cell's energy metabolism. However, the signaling pathways that initiate and regulate activation-induced macroautophagy in T cells have not been identified. Herein, we show that activation-induced macroautophagy in T cells depends on signaling from common γ-chain cytokines. Consequently, inhibition of signaling through JAK3, induced downstream of cytokine receptors containing the common γ-chain, prevents full induction of macroautophagy in activated T cells. Moreover, we found that common γ-chain cytokines are not only required for macroautophagy upregulation during T cell activation but can themselves induce macroautophagy. Our data also show that macroautophagy induction in T cells is associated with an increase of LC3 expression that is mediated by a post-transcriptional mechanism. Overall, our findings unveiled a new role for common γ-chain cytokines as a molecular link between autophagy-induction and T cell activation.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is an essential catabolic process that regulates a diverse array of functions by targeting cellular components for degradation by lysosomes. Studies in mammalian cells have shown that the regulation of autophagy is highly complex and optimization of experimental approaches to analyze this process needs to be developed for each model studied. This chapter provides an overview of two of the most commonly used ways to monitor autophagy activity in T cell. It involves description of common techniques, namely Western blot and cell immunostaining, giving specifi c recommendations for working with T cells and monitoring macroautophagy. We also discuss the analysis required for correct interpretation of the results and quantifi cation of macroautophagy activity.
[Show abstract][Hide abstract] ABSTRACT: Chaperone-mediated autophagy (CMA) targets soluble proteins for lysosomal degradation. Here we found that CMA was activated in T cells in response to engagement of the T cell antigen receptor (TCR), which induced expression of the CMA-related lysosomal receptor LAMP-2A. In activated T cells, CMA targeted the ubiquitin ligase Itch and the calcineurin inhibitor RCAN1 for degradation to maintain activation-induced responses. Consequently, deletion of the gene encoding LAMP-2A in T cells caused deficient in vivo responses to immunization or infection with Listeria monocytogenes. Impaired CMA activity also occurred in T cells with age, which negatively affected their function. Restoration of LAMP-2A in T cells from old mice resulted in enhancement of activation-induced responses. Our findings define a role for CMA in regulating T cell activation through the targeted degradation of negative regulators of T cell activation.
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (Tregs) control autoreactive T cells by inhibiting activation-induced proliferation and cytokine expression. The molecular mechanisms responsible for the inactivation of effector T cells by Tregs remain yet to be fully characterized. We report that T-helper cells stimulated in the presence of Tregs quickly activate NFAT1 and have increased NFAT1-dependent expression of the transcription repressor Ikaros. NFAT1 deficiency or dominant-negative Ikaros compromises Treg-mediated inhibition of T-helper cells in vitro and in vivo. Thus, our results place NFAT-dependent mechanisms as general regulators of T-cell tolerance and show that Treg-mediated suppression of T-helper cells results from the activation of NFAT-regulated gene expression.
[Show abstract][Hide abstract] ABSTRACT: Just when you thought that you had heard it all about autophagy-the conserved cellular process that mediates turnover of cellular constituents in the lysosomes-studies keep coming out highlighting new types of autophagy, new functions for autophagy or even new autophagy-independent roles for the proteins associated with this process. The field of immunology has been riding the autophagic wave since the beginning of its revival; first due to its role in the host defense against pathogens, and more recently through the better understanding of the unique characteristics and functions of different autophagic pathways in immune cells. Here, we describe some of these new functions that are tightening the connection between autophagy and acquired or innate immunity and their malfunctioning with age.
No preview · Article · Jun 2014 · Current Opinion in Immunology
[Show abstract][Hide abstract] ABSTRACT: Tumor cells must overcome apoptosis to survive throughout metastatic dissemination and distal organ colonization. Here we show in the Polyoma Middle T mammary tumor model that N-cadherin expression causes Slug upregulation, which in turn promotes carcinoma cell survival. Slug was dramatically upregulated in metastases relative to primary tumors. Consistent with a role in metastasis, Slug knockdown in carcinoma cells suppressed lung colonization by decreasing cell survival at metastatic sites, but had no effect on tumor cell invasion or extravasation. In support of this idea, Slug inhibition by shRNA, sensitized tumor cells to apoptosis by DNA damage, resulting in caspase-3 and PARP cleavage. The pro-survival effect of Slug was found to be caused by direct repression of the pro-apoptotic gene, Puma, by Slug. Consistent with a pivotal role for a Slug-Puma axis in metastasis, inhibition of Puma by RNA interference in Slug-knockdown cells rescued lung colonization, whereas Puma overexpression in control tumor cells suppressed lung metastasis. The survival function of the Slug-Puma axis was confirmed in human breast cancer cells, where Slug knockdown increased Puma expression and inhibited lung colonization. This study demonstrates a pivotal role for Slug in carcinoma cell survival, implying that disruption of the Slug-Puma axis may impinge on the survival of metastatic cells.
[Show abstract][Hide abstract] ABSTRACT: Just when you thought that you had heard it all about autophagy — the conserved cellular process that mediates turnover of cellular constituents in the lysosomes — studies keep coming out highlighting new types of autophagy, new functions for autophagy or even new autophagy-independent roles for the proteins associated with this process. The field of immunology has been riding the autophagic wave since the beginning of its revival; first due to its role in the host defense against pathogens, and more recently through the better understanding of the unique characteristics and functions of different autophagic pathways in immune cells. Here, we describe some of these new functions that are tightening the connection between autophagy and acquired or innate immunity and their malfunctioning with age.
[Show abstract][Hide abstract] ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
[Show abstract][Hide abstract] ABSTRACT: One of the mechanisms that are in place to control the activation of mature T cells that bear self-reactive antigen receptors is anergy, a long-term state of hyporesponsiveness that is established in T cells in response to suboptimal stimulation. T cells receive signals that result not only from antigen recognition and costimulation but also from other sources, including cytokine receptors, inhibitory receptors or metabolic sensors. Integration of those signals will determine T cell fate. Under conditions that induce anergy, T cells activate a program of gene expression that leads to the production of proteins that block T cell receptor signaling and inhibit cytokine gene expression. In this review we will examine those signals that determine functional outcome following antigen encounter, review current knowledge of the factors that ensure signaling inhibition and epigenetic gene silencing in anergic cells and explore the mechanisms that lead to the reversal of anergy and the reacquisition of effector functions.
No preview · Article · Nov 2013 · Seminars in Immunology
[Show abstract][Hide abstract] ABSTRACT: In response to suboptimal activation, T cells become hyporesponsive, with a severely reduced capacity to proliferate and produce
cytokines upon reencounter with antigen. Chromatin analysis of T cells made tolerant by use of different in vitro and in vivo approaches reveals that the expression of gamma interferon (IFN-γ) is epigenetically silenced in anergic effector TH1 cells.
In those T cells, calcium signaling triggers the expression of Tle4, a member of the Groucho family of corepressors, which
is then recruited to a distal regulatory element in the Ifng locus and causes the establishment of repressive epigenetic marks at the Ifng gene regulatory elements. Consequently, impaired Tle4 activity results in a markedly reduced capacity to inhibit IFN-γ production
in tolerized T cells. We propose that Blimp1-dependent recruitment of Tle4 to the Ifng locus causes epigenetic silencing of the expression of the Ifng gene in anergic TH1 cells. These results define a novel function of Groucho family corepressors in peripheral T cells and
demonstrate that specific mechanisms are activated in tolerant T helper cells to directly repress expression of effector cytokines,
supporting the hypothesis that stable epigenetic imprinting contributes to the maintenance of the tolerance-associated hyporesponsive
phenotype in T cells.
Preview · Article · Nov 2013 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Helper T cells become hyporesponsive in the tumor microenvironment (at least in part) owing to the NFAT1-dependent expression of anergy-associated genes. Anergy constitutes a crucial mechanism to prevent tumor destruction by T cells, and hence may represent a powerful target to boost antitumor immune responses and improve the efficacy of immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Double-negative (DN) thymocytes depend on interleukin 7 (IL-7) for survival. In DN2 and DN3 cells, a new role has now been identified for signaling via the IL-7 receptor and the kinase Jak3 that leads to noncanonical activation of the transcription factor NFATc1 and upregulation of expression of the antiapoptotic protein Bcl-2.
No preview · Article · Jan 2013 · Nature Immunology
[Show abstract][Hide abstract] ABSTRACT: AKT3, a member of the serine/threonine kinase AKT family, is involved in a variety of biologic processes. AKT3 is expressed in immune cells and is the major AKT isoform in the CNS representing 30% of the total AKT expressed in spinal cord, and 50% in the brain. Myelin-oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model in which lymphocytes and monocytes enter the CNS, resulting in inflammation, demyelination, and axonal injury. We hypothesized that during EAE, deletion of AKT3 would negatively affect the CNS of AKT3(-/-) mice, making them more susceptible to CNS damage. During acute EAE, AKT3(-/-)mice were more severely affected than wild type (WT) mice. Evaluation of spinal cords showed that during acute and chronic disease, AKT3(-/-) spinal cords had more demyelination compared with WT spinal cords. Quantitative RT-PCR determined higher levels of IL-2, IL-17, and IFN-γ mRNA in spinal cords from AKT3(-/-) mice than WT. Experiments using bone marrow chimeras demonstrated that AKT3(-/-) mice receiving AKT3-deficient bone marrow cells had elevated clinical scores relative to control WT mice reconstituted with WT cells, indicating that altered function of both CNS cells and bone marrow-derived immune cells contributed to the phenotype. Immunohistochemical analysis revealed decreased numbers of Foxp3(+) regulatory T cells in the spinal cord of AKT3(-/-) mice compared with WT mice, whereas in vitro suppression assays showed that AKT3-deficient Th cells were less susceptible to regulatory T cell-mediated suppression than their WT counterparts. These results indicate that AKT3 signaling contributes to the protection of mice against EAE.
Preview · Article · Jan 2013 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Regulatory T cells (Tregs) play a critical role in maintaining immune tolerance and preventing autoimmune disease. Tregs express the transcription factor Foxp3, which acts as a master regulator of their differentiation and controls their capacity to suppress T cell responses. Tregs have an intrinsically anergic phenotype and do not produce IL-2 or proliferate upon stimulation ex vivo. Recent studies identified that Helios, a member of the Ikaros family of transcription factors, is expressed in Tregs. However, its specific function is not fully understood. In this study, we show that Helios regulates IL-2 production in Tregs by suppressing Il2 gene transcription. Loss of Helios in Tregs breaks their anergic phenotype and results in derepression of the Il2 locus, allowing Tregs to display increased baseline proliferation and to produce IL-2 following stimulation. Conversely, forced expression of Helios in CD4(+)Foxp3(-) T cells results in a loss of their normal ability to produce IL-2. Helios acts by binding to the Il2 promoter and inducing epigenetic modifications that include histone deacetylation. We also show that loss of Helios in Tregs results in decreased Foxp3 binding to the Il2 promoter, indicating that Helios promotes binding of Foxp3 to the Il2 promoter. Interestingly, the loss of Helios in Tregs also causes a decrease in suppressive capacity. Our results identify Helios as a key regulator of Il2 expression in Tregs, contributing to the maintenance of the anergic phenotype.
Preview · Article · Dec 2012 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Lipid modifications are essential in cellular sorting and trafficking inside cells. The role of phosphoinositides in trafficking between Golgi and endocytic/lysosomal compartments has been extensively explored and the kinases responsible for these lipid changes have been identified. In contrast, the mechanisms that mediate exit and recycling from lysosomes (Lys), considered for a long time as terminal compartments, are less understood. In this work, we identify a dynamic association of the lipid kinase PI4KIIIβ with Lys and unveil its regulatory function in lysosomal export and retrieval. We have found that absence of PI4KIIIβ leads to abnormal formation of tubular structures from the lysosomal surface and loss of lysosomal constituents through these tubules. We demonstrate that the kinase activity of PI4KIIIβ is necessary to prevent this unwanted lysosomal efflux under normal conditions, and to facilitate proper sorting when recycling of lysosomal material is needed, such as in the physiological context of lysosomal reformation after prolonged starvation.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a crucial role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins and regulating protein levels in response to extracellular signals. In the last few years specific functions for different forms of autophagy have been identified in many tissues and organs. In the Immune System, autophagy functions range from the elimination infectious agents and the modulation of the inflammatory response, to the selection of antigens for presentation and the regulation of T cell homeostasis and activation. Here, we review the recent advances that have allowed us to better understand why autophagy is a crucial process in the regulation of the innate and adaptive immune responses.
No preview · Article · Oct 2012 · Pharmacological Research
[Show abstract][Hide abstract] ABSTRACT: Anergy is induced in T cells as a consequence of a partial or suboptimal stimulation. Anergic T cells become unresponsive and fail to proliferate and produce cytokines. We had previously shown that in anergic CD4(+) T cells, Ikaros participates in the transcriptional repression of the Il2 gene by recruiting histone deacetylases that cause core histone deacetylation at the Il2 promoter. Here we show that deacetylation at the Il2 promoter is the initial step in a process that leads to the stable silencing of the Il2 gene transcription in anergic T cells. We have found that anergy-induced deacetylation of the Il2 promoter permits binding of the histone methyl-transferase Suv39H1, which trimethylates lysine-9 of histone H3 (Me3H3-K9). Furthermore, the establishment of the Me3H3-K9 mark allows the recruitment of the heterochromatin protein HP1, allowing the silenced Il2 loci to reposition close to heterochromatin-rich regions. Our results indicate that silencing of Il2 transcription in anergic T cells is attained through a series of epigenetic changes that involve the establishment of repressive marks and the subsequent nuclear repositioning of the Il2 loci, which become juxtaposed to transcriptionally silent regions. This mechanism may account for the stable nature of the inhibition of IL-2 production in anergic cells.
Full-text · Article · Sep 2012 · European Journal of Immunology