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

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Interindividual clinical variability in the course of SARS-CoV-2 infection is immense. We report that at least 101 of 987 patients with life-threatening COVID-19 pneumonia had neutralizing IgG auto-Abs against IFN-ω (13 patients), the 13 types of IFN-α (36), or both (52), at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1,227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 were men. A B cell auto-immune phenocopy of inborn errors of type I IFN immunity underlies life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men.

CD8+ cytotoxic T lymphocytes (CTLs) mediate resistance to infectious agents and tumours. Classically, CTLs recognize antigens that are localized in the cytoplasm of target cells, processed and presented as peptide complexes with class I molecules of the major histocompatibility complex (MHC). However, there is evidence for an exogenous pathway whereby antigens that are not expected to gain access to the cytoplasm are presented on MHC class I molecules. The most dramatic example is the in vivo phenomenon of cross-priming: antigens from donor cells are acquired by bone-marrow-derived host antigen-presenting cells (APCs) and presented on MHC class I molecules. Two unanswered questions concern the identity of this bone-marrow-derived cell and how such antigens are acquired. Here we show that human dendritic cells, but not macrophages, efficiently present antigen derived from apoptotic cells, stimulating class I-restricted CD8+ CTLs. Our findings suggest a mechanism by which potent APCs acquire antigens from tumours, transplants, infected cells, or even self-tissue, for stimulation or tolerization of CTLs.

SummaryDendritic cells, but not macrophages, efficiently phagocytose apoptotic cells and cross-present viral, tumor, and self-antigens to CD8 ϩ T cells. This in vitro pathway corresponds to the in vivo phenomena of cross-priming and cross-tolerance. Here, we demonstrate that phagocytosis of apoptotic cells is restricted to the immature stage of dendritic cell (DC) development, and that this process is accompanied by the expression of a unique profile of receptors, in particular the ␣ v ␤ 5 integrin and CD36. Upon maturation, these receptors and, in turn, the phagocytic capacity of DCs, are downmodulated. Macrophages engulf apoptotic cells more efficiently than DCs, and although they express many receptors that mediate this uptake, they lack the ␣ v ␤ 5 integrin. Furthermore, in contrast to DCs, macrophages fail to cross-present antigenic material contained within the engulfed apoptotic cells. Thus, DCs use unique pathways for the phagocytosis, processing, and presentation of antigen derived from apoptotic cells on class I major histocompatibility complex. We suggest that the ␣ v ␤ 5 integrin plays a critical role in the trafficking of exogenous antigen by immature DCs in this cross-priming pathway.

Cell death by necrosis is typically associated with inflammation, in contrast to apoptosis. We have identified additional distinctions between the two types of death that occur at the level of dendritic cells (DCs) and which influence the induction of immunity. DCs must undergo changes termed maturation to act as potent antigen-presenting cells. Here, we investigated whether exposure to apoptotic or necrotic cells affected DC maturation. We found that immature DCs efficiently phagocytose a variety of apoptotic and necrotic tumor cells. However, only exposure to the latter induces maturation. The mature DCs express high levels of the DC-restricted markers CD83 and lysosome-associated membrane glycoprotein (DC-LAMP) and the costimulatory molecules CD40 and CD86. Furthermore, they develop into powerful stimulators of both CD4+ and CD8+ T cells. Cross-presentation of antigens to CD8+ T cells occurs after uptake of apoptotic cells. We demonstrate here that optimal cross-presentation of antigens from tumor cells requires two steps: phagocytosis of apoptotic cells by immature DCs, which provides antigenic peptides for major histocompatibility complex class I and class II presentation, and a maturation signal that is delivered by exposure to necrotic tumor cells, their supernatants, or standard maturation stimuli, e.g., monocyte-conditioned medium. Thus, DCs are able to distinguish two types of tumor cell death, with necrosis providing a control that is critical for the initiation of immunity.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus responsible for a recent, unexpectedly severe epidemic in countries of the Indian Ocean region. Although many alphaviruses have been well studied, little was known about the biology and pathogenesis of CHIKV at the time of the 2005 outbreak. Over the past 5 years there has been a multidisciplinary effort aimed at deciphering the clinical, physiopathological, immunological and virological features of CHIKV infection. This Review highlights some of the most recent advances in our understanding of the biology of CHIKV and its interactions with the host.

Chikungunya virus (CHIKV) is a re-emerging arbovirus responsible for a massive outbreak currently afflicting the Indian Ocean region and India. Infection from CHIKV typically induces a mild disease in humans, characterized by fever, myalgia, arthralgia, and rash. Cases of severe CHIKV infection involving the central nervous system (CNS) have recently been described in neonates as well as in adults with underlying conditions. The pathophysiology of CHIKV infection and the basis for disease severity are unknown. To address these critical issues, we have developed an animal model of CHIKV infection. We show here that whereas wild type (WT) adult mice are resistant to CHIKV infection, WT mouse neonates are susceptible and neonatal disease severity is age-dependent. Adult mice with a partially (IFN-α/βR+/−) or totally (IFN-α/βR−/−) abrogated type-I IFN pathway develop a mild or severe infection, respectively. In mice with a mild infection, after a burst of viral replication in the liver, CHIKV primarily targets muscle, joint, and skin fibroblasts, a cell and tissue tropism similar to that observed in biopsy samples of CHIKV-infected humans. In case of severe infections, CHIKV also disseminates to other tissues including the CNS, where it specifically targets the choroid plexuses and the leptomeninges. Together, these data indicate that CHIKV-associated symptoms match viral tissue and cell tropisms, and demonstrate that the fibroblast is a predominant target cell of CHIKV. These data also identify the neonatal phase and inefficient type-I IFN signaling as risk factors for severe CHIKV-associated disease. The development of a permissive small animal model will expedite the testing of future vaccines and therapeutic candidates.