Gennaro De Libero - Academia.edu (original) (raw)

Papers by Gennaro De Libero

Journal of Experimental Medicine, 2004

Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell respo... more Mycobacterial lipids comprise a heterogeneous group of molecules capable of inducing T cell responses in humans. To identify novel antigenic lipids and increase our understanding of lipid-mediated immune responses, we established a panel of T cell clones with different lipid specificities. Using this approach we characterized a novel lipid antigen belonging to the group of diacylated sulfoglycolipids purified from Mycobacterium tuberculosis. The structure of this sulfoglycolipid was identified as 2-palmitoyl or 2-stearoyl-3-hydroxyphthioceranoyl-2′-sulfate-α-α′-d-trehalose (Ac2SGL). Its immunogenicity is dependent on the presence of the sulfate group and of the two fatty acids. Ac2SGL is mainly presented by CD1b molecules after internalization in a cellular compartment with low pH. Ac2SGL-specific T cells release interferon γ, efficiently recognize M. tuberculosis–infected cells, and kill intracellular bacteria. The presence of Ac2SGL-responsive T cells in vivo is strictly dependent...

Frontiers in immunology, 2018

The definition "unconventional T cells" identifies T lymphocytes that recognize non-pep... more The definition "unconventional T cells" identifies T lymphocytes that recognize non-peptide antigens presented by monomorphic antigen-presenting molecules. Two cell populations recognize lipid antigens and small metabolites presented by CD1 and MR1 molecules, respectively. A third cell population expressing the TCR Vγ9Vδ2 is stimulated by small phosphorylated metabolites. In the recent past, we have learnt a lot about the selection, tissue distribution, gene transcription programs, mode of expansion after antigen recognition, and persistence of these cells. These studies depict their functions in immune homeostasis and diseases. Current investigations are revealing that unconventional T cells include distinct sub-populations, which display unexpected similarities to classical MHC-restricted T cells in terms of TCR repertoire diversity, antigen specificity variety, functional heterogeneity, and naïve-to-memory differentiation dynamic. This review discusses the latest findin...

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Frontiers in immunology, 2017

NLRP10 is a nucleotide-binding oligomerization domain-like receptor that functions as an intracel... more NLRP10 is a nucleotide-binding oligomerization domain-like receptor that functions as an intracellular pattern recognition receptor for microbial products. Here, we generated amouse to delineate the role of NLRP10 in the host immune response and found thatdendritic cells (DCs) elicited sub-optimal IFNγ production by antigen-specific CD4T cells compared to wild-type (WT) DCs. In response to T-cell encounter, CD40 ligation or Toll-like receptor 9 stimulation,DCs produced low levels of IL-12, due to a substantial decrease in NF-κB activation. Defective IL-12 production was also evidentand affected IFNγ production by CD4T cells. Upon() infection,mice displayed diminished T helper 1-cell responses and increased bacterial growth compared to WT mice. These data indicate that NLRP10-mediated IL-12 production by DCs is critical for IFNγ induction in T cells and contributes to promote the host defense against.

Autophagy, 2017

Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functi... more Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4+ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptormediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrindependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4+ T cell stimulation.

Scientific Reports, 2016

Access to point-of-care (POC), rapid, inexpensive, sensitive, and instrument-free tests for the d... more Access to point-of-care (POC), rapid, inexpensive, sensitive, and instrument-free tests for the diagnosis of tuberculosis (TB) remains a major challenge. Here, we report a simple and low-cost microchip-based TB ELISA (MTBE) platform for the detection of anti-mycobacterial IgG in plasma samples in less than 15 minutes. The MTBE employs a flow-less, magnet-actuated, bead-based ELISA for simultaneous detection of IgG responses against multiple mycobacterial antigens. Anti-trehalose 6,6′-dimycolate (TDM) IgG responses were the strongest predictor for differentiating active tuberculosis (ATB) from healthy controls (HC) and latent tuberculosis infections (LTBI). The TDM-based MTBE demonstrated superior sensitivity compared to sputum microscopy (72% vs. 56%) with 80% and 63% positivity among smear-positive and smear-negative confirmed ATB samples, respectively. Receiver operating characteristic analysis indicated good accuracy for differentiating ATB from HC (AUC = 0.77). Thus, TDM-based MTBE can be potentially used as a screening device for rapid diagnosis of active TB at the POC. The management and control of tuberculosis (TB) still remains a significant threat to public health 1 , partly due to the absence of cost-effective, sensitive, and rapid diagnostic tests 2,3. Currently, sputum smear microscopy is the most commonly used point-of-care (POC) method for TB diagnosis in endemic countries, despite its poor sensitivity (30-60%) 4. Although "gold standard" bacterial culture does provide the required sensitivity, the test takes several weeks and requires well-equipped laboratories and trained staff 5. Such a long turnaround time often results in delayed diagnosis, continued transmission, and the risk of developing drug resistance 6. Serological tests based on the detection of antibodies against mycobacterial protein antigens in the form of lateral flow devices or standard ELISAs have been extensively used for the diagnosis of TB 7. However, these tests have demonstrated poor sensitivity (1-60%) and specificity (53-99%) compared with standard culture methods 8 , performing no better than sputum smear microscopy, and have failed to improve patient outcomes. As such, the World Health Organization (WHO) has recommended against their usage 7. Endorsement by the WHO of nucleic acid amplification-based TB diagnostic tests, such as the automated GeneXpert MTB/RIF system (Cepheid), INNO-LiPA Rif TB kit (Innogenetics), and Genotype MTBDRplus assay (Hain Lifescience) has helped to fill this gap. However, their implementation in POC has been severely restricted by high maintenance costs and the need for sophisticated instrumentation, trained personnel, and uninterrupted electrical supply 9. Thus, there is an urgent need for the development of a simple, sensitive, and portable assay for the early stage detection of TB at the POC. An ideal test must meet minimum specifications outlined by the WHO, such as short assay time (< 3 h), minimal sample preparation steps, maintenance-free instrumentation, low-cost (< $10 per test), and environmentally acceptable waste disposability 10,11 .

European Journal of Immunology

Using transgenic mice, we have identified a human CD4 silencer contained within a 484-bp fragment... more Using transgenic mice, we have identified a human CD4 silencer contained within a 484-bp fragment in the first intron of the CD4 gene. Further experiments have mapped a lineage-specific silencing activity to a region of 190 bp. This region contains two protein-binding sites detected by deoxyribonuclease I footprinting analyses. Tested in transient transfection assays, these two DNA elements showed significant silencing activity restricted to the CD8 phenotype. In CD4 cells, either no clear effect (FP I) or strong enhancing activity (FP II) was observed by transient transfection assays. Despite the lineage-specific activity of these two elements, electrophoretic mobility shift assays (EMSA) showed similar levels of protein binding to the silencer element FP I in CD4 and CD8 T cells. Base substitutions in the FP I fragment abolished the silencing activity in transfected CD8 cells as well as the protein binding in EMSA, suggesting an important role of this protein-DNA interaction in CD...

Infectious Agents and Pathogenesis, 1988

Tuberculosis is a bacterial infectious disease that, left untreated, often takes a chronic course... more Tuberculosis is a bacterial infectious disease that, left untreated, often takes a chronic course.1 To a great extent, this chronicity is due to the fact that the etiologic agents, Mycobacterium tuberculosis, M. bovis, and M. africanum have developed means to survive or even grow in one of the host’s most potent effector cells, the mononuclear phagocytes. These cells are particularly specialized to engulf, kill, and degrade invading microorganisms, yet at first sight may not look like an attractive habitat for microbial living. Thus, the evasion mechanisms used by pathogenic mycobacteria must be highly effective. Although these mechanisms have hitherto not been fully elucidated, it is generally believed that they are manifold, including (1) resistance to reactive oxygen metabolites and lysosomal enzymes, (2) inhibition of phagosome-lysosome fusion, and (3) perhaps evasion into the cytoplasm.2

Frontiers in immunology, 2014

T-cells recognize lipid antigens presented by dedicated antigen-presenting molecules that belong ... more T-cells recognize lipid antigens presented by dedicated antigen-presenting molecules that belong to the CD1 family. This review discusses the structural properties of CD1 molecules, the nature of mycobacterial lipid antigens, and the phenotypic and functional properties of T-cells recognizing mycobacterial lipids. In humans, the five CD1 genes encode structurally similar glycoproteins that recycle in and thus survey different cellular endosomal compartments. The structure of the CD1-lipid-binding pockets, their mode of intracellular recycling and the type of CD1-expressing antigen-presenting cells all contribute to diversify lipid immunogenicity and presentation to T-cells. Mycobacteria produce a large variety of lipids, which form stable complexes with CD1 molecules and stimulate specific T-cells. The structures of antigenic lipids may be greatly different from each other and each lipid may induce unique T-cells capable of discriminating small lipid structural changes. The importan...

News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society, 2003

T cells may recognize glycolipids and lipids of bacterial and self origin associated with the CD1... more T cells may recognize glycolipids and lipids of bacterial and self origin associated with the CD1 antigen-presenting molecules. Understanding the mechanisms governing CD1-self glycolipid interaction will provide information on the molecular rules of glycolipid presentation and suggest new approaches to immunotherapy.

Bollettino dell'Istituto sieroterapico milanese, 1983

This study was undertaken in order to investigate the immunomodulatory effect of heat-killed Baci... more This study was undertaken in order to investigate the immunomodulatory effect of heat-killed Bacillus subtilis spores as well as of one soluble and one insoluble fraction obtained after their mechanical breakage. The results demonstrate that heat-killed spores, the soluble and the insoluble fraction enhance the primary PFC response to T-dependent antigens such as SRBC and HRBC, but not to T-independent antigens such as Ficoll-TNP. However, the same doses capable of enhancing the production of antibodies against T-dependent antigens were unable to modify the host CMI, evaluated in terms of contact sensitivity to oxazolone.

Journal of immunology (Baltimore, Md. : 1950), 1988

Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-prim... more Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given toge...

OncoImmunology, 2014

A subset of CD1c-restricted T lymphocytes exhibits strong reactivity against leukemia cells. Thes... more A subset of CD1c-restricted T lymphocytes exhibits strong reactivity against leukemia cells. These T cells recognize methyl-lysophosphatidic acid (mLPA), a novel lipid antigen produced by acute leukemia cells. Considering that CD1crestricted T cells display efficacious anti-leukemia activities in a mouse model, this lipid antigen thus represents a novel target in the immunotherapy of hematological malignancies.

Frontiers in Immunology, 2015

The mechanistic requirements of antigen recognition by T cells expressing a γδ TCR has revealed i... more The mechanistic requirements of antigen recognition by T cells expressing a γδ TCR has revealed important differences with those of αβ TCR cells and, despite impressive new data generated in the very recent years, they remain poorly understood. Based on the structure of the TCR chains and the tissue distribution, γδ cells are represented in a variety of populations. The major subset of human peripheral blood γδ cells express Vγ9Vδ2 TCR heterodimers and are all stimulated by phosphorylated metabolites (commonly called phosphoantigens). Phosphoantigens are molecules with a very small mass and only stimulate Vγ9Vδ2 cells in the presence of antigen-presenting cells, suggesting a strict requirement for dedicated antigen-presenting molecules. Recent studies have identified butyrophilin (BTN) 3A1 as the molecule necessary to stimulate Vγ9Vδ2 cells. BTN3A1 extracellular, transmembrane, and cytoplasmic domains have different functions, including cognate interaction with theVγ9Vδ2TCR, binding of the phosphoantigens, and interaction with cytoplasmic proteins. This review mainly discusses the known molecular mechanisms of BTN3A1-mediated antigen presentation to γδ cells and proposes a model of phosphoantigen presentation, which integrates past and recent studies.

Nature Chemical Biology, 2014

a semisynthetic carbohydrate-lipid vaccine that protects against S. pneumoniae in mice marco cava... more a semisynthetic carbohydrate-lipid vaccine that protects against S. pneumoniae in mice marco cavallari 1,6,7 , pierre stallforth 2,6,7 , artem Kalinichenko 1,6 , dominea c K rathwell 2 , thomas m a gronewold 3 , alexander adibekian 2,7 , lucia mori 1,4 , regine landmann 5 , peter h seeberger 2 & gennaro de libero 1,4 * Severe forms of pneumococcal meningitis, bacteraemia and pneumonia result in more than 1 million deaths each year despite the widespread introduction of carbohydrate-protein conjugate vaccines against Streptococcus pneumoniae. Here we describe a new and highly efficient antipneumococcal vaccine design based on synthetic conjugation of S. pneumoniae capsule polysaccharides to the potent lipid antigen a-galactosylceramide, which stimulates invariant natural killer T (iNKT) cells when presented by the nonpolymorphic antigen-presenting molecule CD1d. Mice injected with the new lipid-carbohydrate conjugate vaccine produced high-affinity IgG antibodies specific for pneumococcal polysaccharides. Vaccination stimulated germinal center formation; accumulation of iNKT cells with a T follicular helper cell phenotype; and increased frequency of carbohydrate-specific, longlived memory B cells and plasmablasts. This new lipid-carbohydrate vaccination strategy induced potent antipolysaccharide immunity that protected against pneumococcal disease in mice and may also prove effective for the design of carbohydratebased vaccines against other major bacterial pathogens.

Methods in Molecular Biology, 2009

Chapter 1: Analysis of Frequency and Phenotype of Antigen-Specific T Cells Angus Stock and Vincen... more Chapter 1: Analysis of Frequency and Phenotype of Antigen-Specific T Cells Angus Stock and Vincenzo Cerundolo Chapter 2: B Cell Helper Assays Sergio Abrignani, Elena Tonti, Giulia Casorati, and Paolo Dellabona Chapter 3: Trans-Kingdom RNA Interference (tkRNAi): A Novel Method to Induce Therapeutic Gene Silencing Thu A. Nguyen and Johannes H. Fruehauf Chapter 4: Flow Cytometry and Cell Activation Sonia Gavasso Chapter 5: Investigating T Cells by Polychromatic Flow Cytometry Enrico Lugli, Leonarda Troiano, and Andrea Cossarizza Chapter 6: Generation of Human T Cell Clones Sabrina Mariotti and Roberto Nisini Chapter 7: Limiting Dilution Analysis of Antigen-Specific T cells Jorge Carneiro, Lurdes Duarte, and Elisabetta Padovan Chapter 8: T Cell Epitope-Mapping by Cytokine Gene Expression Assay Maurizio Provengano and Giulio C. Spagnoli Chapter 9: Cytokine Multiplex Immunoassay: Methodology and (Clinical) Applications Wilco de Jager, Berent Prakken, and Ger T. Rijkers Chapter 10: Purification of the T Cell Antigen Receptor and Analysis by Blue-Native PAGE Mahima Swamy and Wolfgang W.A. Schamel Chapter 11: Non-Replicating Recombinant Vaccinia Virus Expressing CD80 to Enhance T-Cell Stimulation Paul Zajac

Trends in Immunology, 2006

The presentation of lipid antigens by CD1 molecules follows precise rules imposed by the biochemi... more The presentation of lipid antigens by CD1 molecules follows precise rules imposed by the biochemical nature of lipids. The structures of CD1-lipid complexes are elucidating how T-cell receptors interact with hydrophobic antigens. The mechanism of lipid uptake and the pathways followed by lipids embedded in the cell membrane contribute to the efficient presentation of exogenous and self-lipids. Lipid presentation is further regulated by the trafficking route of CD1 proteins and their precise membrane localization within endosomal vesicles. Moreover, the generation of immunogenic lipids might require adequate processing, which occurs in the presence of lipid-binding proteins, including CD1e. Here, we review recent experimental evidence that has revealed new protagonists involved in generating immunogenic lipids and has indicated unexpected biological mechanisms contributing to immune recognition.

Tetrahedron, 2002

Lipids U 0750 CD1a-Binding Glycosphingolipids Stimulating Human Autoreactive T-Cells: Synthesis o... more Lipids U 0750 CD1a-Binding Glycosphingolipids Stimulating Human Autoreactive T-Cells: Synthesis of a Family of Sulfatides Differing in the Acyl Chain Moiety.-Title compounds like (I) are capable of stimulating sulfatide-specific and CD1a-restricted T-cell clones.

Science, 1993

Lymphocytes recognize antigens with highly variable heterodimeric surface receptors. Although fou... more Lymphocytes recognize antigens with highly variable heterodimeric surface receptors. Although four distinct antigen receptors could in principle be produced by any lymphocyte, only one functional combination of receptor chains has thus far been found expressed on their surface. Examination of human gamma delta T cells revealed a population that violated this rule by expressing on their surface two distinct functional gamma delta T cell receptors (TCRs) that used different TCR gamma gene alleles. Thus, current models for T cell clonal selection may need modification, and a possible escape mechanism for autoreactive TCRs is suggested.

Proceedings of the National Academy of Sciences, 2001

Proceedings of the National Academy of Sciences, 1994

y8 T cells respond to a variety of microbial pathogens and transormed cells. Their limited recept... more y8 T cells respond to a variety of microbial pathogens and transormed cells. Their limited receptor repertoire and activation by mycobacterial antigens resistant to proteases suggest that they may recognize nonpeptide antigens. We have tested a variety of nonpeptide molecules for stimulation of human y8 T cells. Synthetic alkyl phosphates, particularly monoethyl phosphate (MEP), selectively activated yS T