Centre d’Immunologie de Marseille-Luminy

The Carbohydrate-Active Enzymes database (CAZy; http://www.cazy.org) provides online and continuously updated access to a sequence-based family classification linking the sequence to the specificity and 3D structure of the enzymes that assemble, modify and breakdown oligo- and polysaccharides. Functional and 3D structural information is added and curated on a regular basis based on the available literature. In addition to the use of the database by enzymologists seeking curated information on CAZymes, the dissemination of a stable nomenclature for these enzymes is probably a major contribution of CAZy. The past few years have seen the expansion of the CAZy classification scheme to new families, the development of subfamilies in several families and the power of CAZy for the analysis of genomes and metagenomes. This article outlines the changes that have occurred in CAZy during the past 5 years and presents our novel effort to display the resolution and the carbohydrate ligands in crystallographic complexes of CAZymes.

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 thatthere 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 completeprocess 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 increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 manycases, 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 forreviewers 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 multipleassays 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 autophagyrelated 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.

The nuclear factor NF-kappaB pathway has long been considered a prototypical proinflammatory signaling pathway, largely based on the role of NF-kappaB in the expression of proinflammatory genes including cytokines, chemokines, and adhesion molecules. In this article, we describe how genetic evidence in mice has revealed complex roles for the NF-kappaB in inflammation that suggest both pro- and anti-inflammatory roles for this pathway. NF-kappaB has long been considered the "holy grail" as a target for new anti-inflammatory drugs; however, these recent studies suggest this pathway may prove a difficult target in the treatment of chronic disease. In this article, we discuss the role of NF-kappaB in inflammation in light of these recent studies.

Monocytes and macrophages are critical effectors and regulators of inflammation and the innate immune response, the immediate arm of the immune system. Dendritic cells initiate and regulate the highly pathogen-specific adaptive immune responses and are central to the development of immunologic memory and tolerance. Recent in vivo experimental approaches in the mouse have unveiled new aspects of the developmental and lineage relationships among these cell populations. Despite this, the origin and differentiation cues for many tissue macrophages, monocytes, and dendritic cell subsets in mice, and the corresponding cell populations in humans, remain to be elucidated.

Natural killer (NK) cells were originally defined as effector lymphocytes of innate immunity endowed with constitutive cytolytic functions. More recently, a more nuanced view of NK cells has emerged. NK cells are now recognized to express a repertoire of activating and inhibitory receptors that is calibrated to ensure self-tolerance while allowing efficacy against assaults such as viral infection and tumor development. Moreover, NK cells do not react in an invariant manner but rather adapt to their environment. Finally, recent studies have unveiled that NK cells can also mount a form of antigen-specific immunologic memory. NK cells thus exert sophisticated biological functions that are attributes of both innate and adaptive immunity, blurring the functional borders between these two arms of the immune response.

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Monocytes and cells of the dendritic cell lineage circulate in blood and eventually migrate into tissue where they further mature and serve various functions, most notably in immune defense. Over recent years these cells have been characterized in detail with the use of cell surface markers and flow cytometry, and subpopulations have been described. The present document proposes a nomenclature for these cells and defines 3 types of monocytes (classical, intermediate, and nonclassical monocytes) and 3 types of dendritic cells (plasmacytoid and 2 types of myeloid dendritic cells) in human and in mouse blood. This classification has been approved by the Nomenclature Committee of the International Union of Immunological Societies, and we are convinced that it will facilitate communication among experts and in the wider scientific community.

Cyclophosphamide is one of several clinically important cancer drugs whose therapeutic efficacy is due in part to their ability to stimulate antitumor immune responses. Studying mouse models, we demonstrate that cyclophosphamide alters the composition of microbiota in the small intestine and induces the translocation of selected species of Gram-positive bacteria into secondary lymphoid organs. There, these bacteria stimulate the generation of a specific subset of "pathogenic" T helper 17 (pT(H)17) cells and memory T(H)1 immune responses. Tumor-bearing mice that were germ-free or that had been treated with antibiotics to kill Gram-positive bacteria showed a reduction in pT(H)17 responses, and their tumors were resistant to cyclophosphamide. Adoptive transfer of pT(H)17 cells partially restored the antitumor efficacy of cyclophosphamide. These results suggest that the gut microbiota help shape the anticancer immune response.

Viral infection of mammalian host results in the activation of innate immune responses. Toll-like receptors (TLRs) have been shown to mediate the recognition of many types of pathogens, including viruses. The genomes of viruses possess unique characteristics that are not found in mammalian genomes, such as high CpG content and double-stranded RNA. These genomic nucleic acids serve as molecular signatures associated with viral infections. Here we show that TLR7 recognizes the single-stranded RNA viruses, vesicular stomatitis virus and influenza virus. The recognition of these viruses by plasmacytoid dendritic cells and B cells through TLR7 results in their activation of costimulatory molecules and production of cytokines. Moreover, this recognition required intact endocytic pathways. Mice deficient in either the TLR7 or the TLR adaptor protein MyD88 demonstrated reduced responses to in vivo infection with vesicular stomatitis virus. These results demonstrate microbial ligand recognition by TLR7 and provide insights into the pathways used by the innate immune cells in the recognition of viral pathogens.

Monocytes are circulating blood leukocytes that play important roles in the inflammatory response, which is essential for the innate response to pathogens. But inflammation and monocytes are also involved in the pathogenesis of inflammatory diseases, including atherosclerosis. In adult mice, monocytes originate in the bone marrow in a Csf-1R (MCSF-R, CD115)-dependent manner from a hematopoietic precursor common for monocytes and several subsets of macrophages and dendritic cells (DCs). Monocyte heterogeneity has long been recognized, but in recent years investigators have identified three functional subsets of human monocytes and two subsets of mouse monocytes that exert specific roles in homeostasis and inflammation in vivo, reminiscent of those of the previously described classically and alternatively activated macrophages. Functional characterization of monocytes is in progress in humans and rodents and will provide a better understanding of the pathophysiology of inflammation.

Two molecular mechanisms of T cell-mediated cytotoxicity, one perforin-based, the other Fas-based, have been demonstrated. To determine the extent of their contribution to T cell-mediated cytotoxicity, a range of effector cells from normal control or perforin-deficient mice were tested against a panel of target cells with various levels of Fas expression. All cytotoxicity observed was due to either of these mechanisms, and no third mechanism was detected. Thus, the perforin- and Fas-based mechanisms may account for all T cell-mediated cytotoxicity in short-term in vitro assays.

The transport of specific molecules across lipid membranes is an essential function of all living organisms and a large number of specific transporters have evolved to carry out this function. The largest transporter gene family is the ATP-binding cassette (ABC) transporter superfamily. These proteins translocate a wide variety of substrates including sugars, amino acids, metal ions, peptides, and proteins, and a large number of hydrophobic compounds and metabolites across extra- and intracellular membranes. ABC genes are essential for many processes in the cell, and mutations in these genes cause or contribute to several human genetic disorders including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response. Characterization of eukaryotic genomes has allowed the complete identification of all the ABC genes in the yeast Saccharomyces cerevisiae, Drosophila, and C. elegans genomes. To date, there are 48 characterized human ABC genes. The genes can be divided into seven distinct subfamilies, based on organization of domains and amino acid homology. Many ABC genes play a role in the maintenance of the lipid bilayer and in the transport of fatty acids and sterols within the body. Here, we review the current knowledge of the human ABC genes, their role in inherited disease, and understanding of the topology of these genes within the membrane.

Monoclonal antibody GK1.5 recognizes a previously undescribed murine T cell surface molecule, designated L3T4, which migrates on SDS-PAGE under reducing conditions as a single band with an apparent m.w. of 52,000. L3T4 is expressed by approximately 80% of thymocytes and by approximately 20% of spleen cells. There appears to be poor correlation between expression of L3T4 by functional T cell clones and expression of Lyt-2, expression of the cytolytic phenotype, and class I MHC antigen reactivity. On the other hand, both a class II MHC antigen-reactive HTL clone and an Lyt-1- Mls-reactive HTL clone express L3T4. Analysis of the effect of mAb GK1.5 on PFC responses in adoptive transfer suggests that L3T4 is expressed by the helper/inducer subset of murine T cells. Expression of L3T4 by murine T cells, however, may correlate primarily with class II MHC antigen reactivity rather than with functional phenotype; mAb GK1.5 profoundly blocks antigen-specific cytolysis by the cloned class II MHC antigen-reactive CTL line A15-1.17. Antigen-specific cytolysis by A15-1.17 is blocked by mAb GK1.5 at a step before the lethal hit. Collectively, the flow cytometric, functional, and biochemical data indicate that L3T4 is similar to the human Leu-3/T4 molecule.

For survival, embryonic motoneurons in vertebrates depend on as yet undefined neurotrophic factors present in the limb bud. Members of the neurotrophin family are currently the best candidates for such neurotrophic factors, but inactivation of their receptor genes leads to only partial loss of motoneurons, which suggests that other factors are involved. Glial cell line-derived neurotrophic factor (GDNF), originally identified as a trophic factor specific for dopaminergic neurons, was found to be 75-fold more potent than the neurotrophins in supporting the survival of purified embryonic rat motoneurons in culture. GDNF messenger RNA was found in the immediate vicinity of motoneurons during the period of cell death in development. In vivo, GDNF rescues and prevents the atrophy of facial motoneurons that have been deprived of target-derived survival factors by axotomy. GDNF may therefore be a physiological trophic factor for spinal motoneurons. Its potency and specificity in vitro and in vivo also make it a good candidate for treatment of motoneuron disease.

Tissue-resident macrophages can develop from circulating adult monocytes or from primitive yolk sac-derived macrophages. The precise ontogeny of alveolar macrophages (AMFs) is unknown. By performing BrdU labeling and parabiosis experiments in adult mice, we found that circulating monocytes contributed minimally to the steady-state AMF pool. Mature AMFs were undetectable before birth and only fully colonized the alveolar space by 3 d after birth. Before birth, F4/80(hi)CD11b(lo) primitive macrophages and Ly6C(hi)CD11b(hi) fetal monocytes sequentially colonized the developing lung around E12.5 and E16.5, respectively. The first signs of AMF differentiation appeared around the saccular stage of lung development (E18.5). Adoptive transfer identified fetal monocytes, and not primitive macrophages, as the main precursors of AMFs. Fetal monocytes transferred to the lung of neonatal mice acquired an AMF phenotype via defined developmental stages over the course of one week, and persisted for at least three months. Early AMF commitment from fetal monocytes was absent in GM-CSF-deficient mice, whereas short-term perinatal intrapulmonary GM-CSF therapy rescued AMF development for weeks, although the resulting AMFs displayed an immature phenotype. This demonstrates that tissue-resident macrophages can also develop from fetal monocytes that adopt a stable phenotype shortly after birth in response to instructive cytokines, and then self-maintain throughout life.

Microglia, the resident myeloid cells of the central nervous system, play important roles in life-long brain maintenance and in pathology. Despite their importance, their regulatory dynamics during brain development have not been fully elucidated. Using genome-wide chromatin and expression profiling coupled with single-cell transcriptomic analysis throughout development, we found that microglia undergo three temporal stages of development in synchrony with the brain--early, pre-, and adult microglia--which are under distinct regulatory circuits. Knockout of the gene encoding the adult microglia transcription factor MAFB and environmental perturbations, such as those affecting the microbiome or prenatal immune activation, led to disruption of developmental genes and immune response pathways. Together, our work identifies a stepwise microglia developmental program integrating immune response pathways that may be associated with several neurodevelopmental disorders.

T cell function in combination with PD-x axis blockade. Monalizumab also stimulated NK cell activity against antibody-coated target cells. Interim results of a phase II trial of monalizumab plus cetuximab in previously treated squamous cell carcinoma of the head and neck showed a 31% objective response rate. Most common adverse events were fatigue (17%), pyrexia (13%), and headache (10%). NKG2A targeting with monalizumab is thus a novel checkpoint inhibitory mechanism promoting anti-tumor immunity by enhancing the activity of both T and NK cells, which may complement first-generation immunotherapies against cancer.

Some Toll and Toll-like receptors (TLRs) provide immunity to experimental infections in animal models, but their contribution to host defense in natural ecosystems is unknown. We report a dominant-negative TLR3 allele in otherwise healthy children with herpes simplex virus 1 (HSV-1) encephalitis. TLR3 is expressed in the central nervous system (CNS), where it is required to control HSV-1, which spreads from the epithelium to the CNS via cranial nerves. TLR3 is also expressed in epithelial and dendritic cells, which apparently use TLR3-independent pathways to prevent further dissemination of HSV-1 and to provide resistance to other pathogens in TLR3-deficient patients. Human TLR3 appears to be redundant in host defense to most microbes but is vital for natural immunity to HSV-1 in the CNS, which suggests that neurotropic viruses have contributed to the evolutionary maintenance of TLR3.

Tissue macrophages have a split personality Resident tissue macrophages (RTMs) reside in various tissue-specific niches during development. They evince microenvironment-directed phenotypes that support host defense and tissue homeostasis. Chakarov et al. used single-cell RNA sequencing and fate-mapping of murine lung RTMs to interrogate RTM-subset heterogeneity, interrelationships, and ontogeny (see the Perspective by Mildner and Yona). In addition to alveolar macrophages, they identified two different interstitial macrophage populations. One population mostly abutted nerve fibers; the other population preferentially localized near blood vessels and appeared to support vessel integrity and inhibit inflammatory cell infiltration into tissues. Science , this issue p. eaau0964 ; see also p. 1154

We describe here the properties of mAb GK1.5, which recognizes a cell surface molecule designated L3T4; the determinant on L3T4 recognized by mAb GK1.5 is designated L3T4a. We present evidence here that: i) the expression of L3T4a by murine T cell clones correlates primarily with class II MHC antigen-reactivity; ii) mAb GK1.5 blocks all class II MHC antigen-specific functions (cytolysis, proliferation, release of lymphokines) by murine class II MHC antigen-reactive T cell clones, although there appears to be clonal heterogeneity in the degree to which these functions are blocked by mAb GK1.5; iii) mAb GK1.5 blocks class II MHC antigen-specific release of IL-2 from cloned T cell hybridomas by blocking class II MHC antigen-specific binding; and iv) L3T4 is very similar to the human Leu3/T4 antigen. The properties of mAb GK1.5 (complement fixation, reactivity with all mouse strains tested, profound blocking of all class II MHC antigen-specific functions by murine T cells, usefulness for FACS analyses, and usefulness for immuno-precipitation/SDS-PAGE analyses) make it suitable for investigating both the role of class II MHC antigen-reactive T cells in various immunological phenomena and the mechanistic basis, at the molecular level, of class II MHC antigen-reactivity by murine T cells.