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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field

[Abstract unavailable

Hypoxia selectively up-regulates PD-L1 on myeloid-derived suppressor cells via HIF-1a, thus affecting T cell activation.

-The tumor microenvironment is a complex system, playing an important role in tumor development and progression. Besides cellular stromal components, extracellular matrix fibers, cytokines, and other metabolic mediators are also involved. In this review we outline the potential role of hypoxia, a major feature of most solid tumors, within the tumor microenvironment and how it contributes to immune resistance and immune suppression/tolerance and can be detrimental to antitumor effector cell functions. We also outline how hypoxic stress influences immunosuppressive pathways involving macrophages, myeloid-derived suppressor cells, T regulatory cells, and immune checkpoints and how it may confer tumor resistance. Finally, we discuss how microenvironmental hypoxia poses both obstacles and opportunities for new therapeutic immune interventions.hypoxia; hypoxia-inducible factor; tumor microenvironment; myeloid cells; lymphoid cells; immune suppression; cancer stem cells; programmed death-ligand 1; epithelial-mesenchymal transition; circulating tumor cells; autophagy and antitumor immune response IT HAS BECOME INCREASINGLY apparent that malignant cells exist in a complex cellular and extracellular microenvironment that plays key roles in the initiation and maintenance of the malignant phenotype. Among the microenvironmental factors that play a dominant role in neoplasia, hypoxia is believed to be one of the most relevant in the neoplastic response of tumor cells. It is widely appreciated that the majority of malignancies create a hostile hypoxic microenvironment that can hamper cellmediated immunity and dampen the efficacy of the immune response. Poorly vascularized and hypoxic zones inside solid tumors contribute to immune tolerance of tumor cells by impeding the homing of immunocompetent cells into tumors and inhibiting their antitumor efficacy. Several regulatory mechanisms can occur concurrently within the hypoxic tumor microenvironment, resulting in multiple redundant levels of immune cell plasticity and suppression, tumor plasticity, and functional heterogeneity. Hypoxic stress clearly plays a crucial role in tumor promotion and immune escape by controlling angiogenesis and favoring immune suppression and tumor resistance. Like other therapeutic attempts, immunotherapy is heavily hampered by the morphologically aberrant tumor microvasculature, preventing migration of immune effector cells into established tumors. Many strategies are emerging for changing the immunosuppressive nature of the tumor to a microenvironment able to support antitumor immunity. The identification of ways to induce a permissive and less hostile tumor microenvironment to avoid tumor resistance and immune suppression is of major interest and pertinence. Hypoxia as an Integral Component of the Tumor MicroenvironmentAll life on Earth depends on O 2 , and all physiological and pathological processes of a living cell rely principally on O 2 (103). Hypoxia is characterized by lack of O 2 , and hypoxic tissues are inadequately oxygenated (107). A p...

Novel immunotherapy approaches have provided durable remission in a significant number of cancer patients with cancers previously considered rapidly lethal. Nonetheless, the high degree of nonresponders, and in some cases the emergence of resistance in patients who do initially respond, represents a significant challenge in the field of cancer immunotherapy. These issues prompt much more extensive studies to better understand how cancer cells escape immune surveillance and resist immune attacks. Here, we review the current knowledge of how cellular heterogeneity and plasticity could be involved in shaping the tumor microenvironment (TME) and in controlling antitumor immunity. Indeed, recent findings have led to increased interest in the mechanisms by which cancer cells undergoing epithelial‐mesenchymal transition (EMT), or oscillating within the EMT spectrum, might contribute to immune escape through multiple routes. This includes shaping of the TME and decreased susceptibility to immune effector cells. Although much remains to be learned on the mechanisms at play, cancer cell clones with mesenchymal features emerging from the TME seem to be primed to face immune attacks by specialized killer cells of the immune system, the natural killer cells, and the cytotoxic T lymphocytes. Recent studies investigating patient tumors have suggested EMT as a candidate predictive marker to be explored for immunotherapy outcome. Promising data also exist on the potential utility of targeting these cancer cell populations to at least partly overcome such resistance. Research is now underway which may lead to considerable progress in optimization of treatments.

Among cells present in the tumor microenvironment, activated fibroblasts termed cancer-associated fibroblasts (CAFs), play a critical role in the complex process of tumor-stroma interaction. CAFs, one of the prominent stromal cell populations in most types of human carcinomas, have been involved in tumor growth, angiogenesis, cancer stemness, extracellular matrix remodeling, tissue invasion, metastasis, and even chemoresistance. During the past decade, these activated tumor-associated fibroblasts have also been involved in the modulation of the anti-tumor immune response on various levels. In this review, we describe our current understanding of how CAFs accomplish this task as well as their potential therapeutic implications.

Various T cell adhesion molecules and their cognate receptors on target cells promote T cell receptor (TCR)–mediated cell killing. In this report, we demonstrate that the interaction of epithelial cell marker E-cadherin with integrin αE(CD103)β7, often expressed by tumor-infiltrating lymphocytes (TILs), plays a major role in effective tumor cell lysis. Indeed, we found that although tumor-specific CD103+ TIL-derived cytotoxic T lymphocyte (CTL) clones are able to kill E-cadherin+/intercellular adhesion molecule 1− autologous tumor cells, CD103− peripheral blood lymphocyte (PBL)-derived counterparts are inefficient. This cell killing is abrogated after treatment of the TIL clones with a blocking anti-CD103 monoclonal antibody or after targeting E-cadherin in the tumor using ribonucleic acid interference. Confocal microscopy analysis also demonstrated that αEβ7 is recruited at the immunological synapse and that its interaction with E-cadherin is required for cytolytic granule polarization and subsequent exocytosis. Moreover, we report that the CD103− profile, frequently observed in PBL-derived CTL clones and associated with poor cytotoxicity against the cognate tumor, is up-regulated upon TCR engagement and transforming growth factor β1 treatment, resulting in strong potentiation of antitumor lytic function. Thus, CD8+/CD103+ tumor-reactive T lymphocytes infiltrating epithelial tumors most likely play a major role in antitumor cytotoxic response through αEβ7–E-cadherin interactions.

Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies.hypoxic tumor microenvironment | innate immunity | breast adenocarcinoma | immunotherapy