Giorgio Napolitani

Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells.

Interleukin 17 (IL-17)–producing CD4+ helper T cells (TH-17 cells) have been linked to host defense and autoimmune diseases. In mice, the differentiation of TH-17 cells requires transforming growth factor-β and IL-6 and the transcription factor RORγt. We report here that for human naive CD4+ T cells, RORγt expression and TH-17 polarization were induced by IL-1β and enhanced by IL-6 but were suppressed by transforming growth factor-β and IL-12. Monocytes and conventional dendritic cells, but not monocyte-derived dendritic cells activated by microbial stimuli, efficiently induced TH-17 priming, and this function correlated with antigen-presenting cell production of IL-1β and IL-6 but not IL-12. Our results identify cytokines, antigen-presenting cells and microbial products that promote the polarization of human TH-17 cells and emphasize an important difference in the requirements for the differentiation of TH-17 cells in humans and mice.

Surface phenotype and antigenic specificity of human interleukin 17–producing T helper memory cells

Interleukin 17 (IL-17)–producing T helper cells (TH-17 cells) have been characterized in mice as a distinct subset of effector cells, but their identity and properties in humans remain elusive. We report here that expression of CCR6 and CCR4 together identified human memory CD4+ T cells selectively producing IL-17 and expressing mRNA encoding the human ortholog of mouse RORγt, a transcription factor, whereas CCR6 and CXCR3 identified TH1 cells producing interferon-γ and T helper cells producing both interferon-γ and IL-17. Memory T cells specific for Candida albicans were present mainly in the CCR6+CCR4+ TH-17 subset, whereas memory T cells specific for Mycobacterium tuberculosis were present in CCR6+CXCR3+ T helper type 1 subset. The elicitation of IL-17 responses correlated with the capacity of C. albicans hyphae to stimulate antigen-presenting cells for the priming of TH-17 responses in vitro and for the production of IL-23 but not IL-12. Our results demonstrate that human TH-17 cells have distinct migratory capacity and antigenic specificities and establish a link between microbial products, T helper cell differentiation and homing in response to fungal antigens.

Prostaglandin E2 enhances Th17 responses via modulation of IL-17 and IFN-γ production by memory CD4+ T cells

The contribution of Th1 and Th17 cells in chronic inflammatory conditions leading to autoimmunity remains highly controversial. In inflamed tissues, production of prostaglandins by COX‐2 has been proposed to favor Th17 responses indirectly by increasing IL‐23 and blocking IL‐12 release from APC. We report here that prostaglandin E2 (PGE2) can directly modulate cytokine production by human memory CD4+ T cells. TCR triggering in the presence of PGE2 increased IL‐17 and reduced IFN‐γ production by freshly isolated memory T cells or T‐cell clones. PGE2 triggered the EP2 and EP4 receptors expressed on T cells leading to a rapid increase of retinoic‐acid‐related orphan receptor‐γt (ROR‐γt) and decrease of T‐cell‐specific T‐box transcription factor 21 (T‐bet) mRNA. Moreover, PGE2 promoted the selective enrichment of IL‐17‐producing cells by differentially modulating the proliferation of memory T‐cell subsets in vitro. Taken together our results indicate that T‐cell effector function is a direct target for PGE2 modulation and suggest a novel mechanism by which inhibitors of prostaglandin synthesis, such as COX‐2 inhibitors, exert their anti‐inflammatory effect.

Simvastatin inhibits the MHC class II pathway of antigen presentation by impairing Ras superfamily GTPases

Statins are widely used hypocholesterolemic drugs that inhibit 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase, a rate‐limiting enzyme of the mevalonate pathway whose biosynthetic endproduct is cholesterol. As a result of this activity, statins may perturb the composition of cell membranes, resulting in lipid raft disruption. Furthermore, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and activity of small GTPases. Antigen uptake, processing and presentation involve the interplay of Rab and Rho family GTPases. Furthermore, lipid rafts have been implicated both in antigen internalization by the BCR and in MHC class II clustering at the immunological synapse. Here we have addressed the effects of simvastatin on antigen processing and presentation by human B cells and dendritic cells. The results show that simvastatin potently suppresses tetanus toxoid processing and presentation to CD4+ T cells by HLA‐DR by inhibiting protein antigen uptake through both receptor‐mediated endocytosis and macropinocytosis. This effect can be largely accounted for by defective prenylation of Rho and Rab GTPases in the absence of any measurable perturbation of lipid rafts. In addition, simvastatin was found to preferentially affect the invariant chain‐dependent MHC class II pathway, thereby identifying this route of antigen processing and presentation as a selective target of statins.

Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

Significance Understanding how to optimize lipid-loading onto CD1d molecules is important to better harness invariant natural killer T (iNKT) cells’ central role at the interface between innate and adaptive immunity. We report that the lipid transfer proteins saposins play an essential role in modulating human iNKT cell autoreactivity to antigen-presenting cells activated by inflammatory stimuli. Lipid-loading occurs in an endo-lysosomal compartment, where saposins work as “lipid editors,” capable of fine-tuning loading and unloading of CD1d molecules and increasing the off-rate of CD1d-bound lipids. Lipid transfer proteins, such as molecules of the saposin family, facilitate extraction of lipids from biological membranes for their loading onto CD1d molecules. Although it has been shown that prosaposin-deficient mice fail to positively select invariant natural killer T (iNKT) cells, it remains unclear whether saposins can facilitate loading of endogenous iNKT cell agonists in the periphery during inflammatory responses. In addition, it is unclear whether saposins, in addition to loading, also promote dissociation of lipids bound to CD1d molecules. To address these questions, we used a combination of cellular assays and demonstrated that saposins influence CD1d-restricted presentation to human iNKT cells not only of exogenous lipids but also of endogenous ligands, such as the self-glycosphingolipid β-glucopyranosylceramide, up-regulated by antigen-presenting cells following bacterial infection. Furthermore, we demonstrated that in human myeloid cells CD1d-loading of endogenous lipids after bacterial infection, but not at steady state, requires trafficking of CD1d molecules through an endo-lysosomal compartment. Finally, using BIAcore assays we demonstrated that lipid-loaded saposin B increases the off-rate of lipids bound to CD1d molecules, providing important insights into the mechanisms by which it acts as a “lipid editor,” capable of fine-tuning loading and unloading of CD1d molecules. These results have important implications in understanding how to optimize lipid-loading onto antigen-presenting cells, to better harness iNKT cells central role at the interface between innate and adaptive immunity.

Intestinal DC in migrational imprinting of immune cells.

Dendritic cells (DCs) have a pivotal role in instructing antigen‐specific immune responses, processing and presenting antigens to CD4+ and CD8+ T cells and producing factors capable to modulate the quality of T‐cell responses. In this review, we will provide an historic overview on the identification of the mechanisms controlling lymphocyte migration into the largest immune organ of the body: the gut, and we will describe how in recent years an unexpected role for DCs has emerged as the architects in programming gut‐homing immune cells. Specifically, we will review how intestinal DCs utilize the dietary vitamin A metabolite retinoic acid (RA) to program gut‐homing lymphocytes and how intestinal DCs acquire the unique capacity to become RA producers.

Activation of Human Mucosal-Associated Invariant T Cells Induces CD40L-Dependent Maturation of Monocyte-Derived and Primary Dendritic Cells

Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize intermediates of the vitamin B2 biosynthetic pathway presented by the monomorphic MR1 molecule. It remains unclear whether, in addition to their cytolytic activity that is important in antimicrobial defense, MAIT cells have immune-modulatory functions that could enhance dendritic cell (DC) maturation. In this study, we investigated the molecular mechanisms dictating the interactions between human MAIT cells and DCs and demonstrate that human MAIT cells mature monocyte-derived and primary DCs in an MR1- and CD40L-dependent manner. Furthermore, we show that MAIT cell–derived signals synergize with microbial stimuli to induce secretion of bioactive IL-12 by DCs. Activation of human MAIT cells in whole blood leads to MR1- and cytokine-dependent NK cell transactivation. Our results underscore an important property of MAIT cells, which can be of translational relevance to rapidly orchestrate adaptive immunity through DC maturation.

AS03- and MF59-Adjuvanted Influenza Vaccines in Children

Influenza is a major cause of respiratory disease leading to hospitalization in young children. However, seasonal trivalent influenza vaccines (TIVs) have been shown to be ineffective and poorly immunogenic in this population. The development of live-attenuated influenza vaccines and adjuvanted vaccines are important advances in the prevention of influenza in young children. The oil-in-water emulsions MF59 and adjuvant systems 03 (AS03) have been used as adjuvants in both seasonal adjuvanted trivalent influenza vaccines (ATIVs) and pandemic monovalent influenza vaccines. Compared with non-adjuvanted vaccine responses, these vaccines induce a more robust and persistent antibody response for both homologous and heterologous influenza strains in infants and young children. Evidence of a significant improvement in vaccine efficacy with these adjuvanted vaccines resulted in the use of the monovalent (A/H1N1) AS03-adjuvanted vaccine in children in the 2009 influenza pandemic and the licensure of the seasonal MF59 ATIV for children aged 6 months to 2 years in Canada. The mechanism of action of MF59 and AS03 remains unclear. Adjuvants such as MF59 induce proinflammatory cytokines and chemokines, including CXCL10, but independently of type-1 interferon. This proinflammatory response is associated with improved recruitment, activation and maturation of antigen presenting cells at the injection site. In young children MF59 ATIV produced more homogenous and robust transcriptional responses, more similar to adult-like patterns, than did TIV. Early gene signatures characteristic of the innate immune response, which correlated with antibody titers were also identified. Differences were detected when comparing child and adult responses including opposite trends in gene set enrichment at day 3 postvaccination and, unlike adult data, a lack of correlation between magnitude of plasmablast response at day 7 and antibody titers at day 28 in children. These insights show the utility of novel approaches in understanding new adjuvants and their importance for developing improved influenza vaccines for children.

Active nuclear transcriptome analysis reveals inflammasome-dependent mechanism for early neutrophil response to Mycobacterium marinum

The mechanisms governing neutrophil response to Mycobacterium tuberculosis remain poorly understood. In this study we utilise biotagging, a novel genome-wide profiling approach based on cell type-specific in vivo biotinylation in zebrafish to analyse the initial response of neutrophils to Mycobacterium marinum, a close genetic relative of M. tuberculosis used to model tuberculosis. Differential expression analysis following nuclear RNA-seq of neutrophil active transcriptomes reveals a significant upregulation in both damage-sensing and effector components of the inflammasome, including caspase b, NLRC3 ortholog (wu: fb15h11) and il1β. Crispr/Cas9-mediated knockout of caspase b, which acts by proteolytic processing of il1β, results in increased bacterial burden and less infiltration of macrophages to sites of mycobacterial infection, thus impairing granuloma development. We also show that a number of immediate early response genes (IEGs) are responsible for orchestrating the initial neutrophil response to mycobacterial infection. Further perturbation of the IEGs exposes egr3 as a key transcriptional regulator controlling il1β transcription.

Generation of a double binary transgenic zebrafish model to study myeloid gene regulation in response to oncogene activation in melanocytes

ABSTRACT A complex network of inflammatory genes is closely linked to somatic cell transformation and malignant disease. Immune cells and their associated molecules are responsible for detecting and eliminating cancer cells as they establish themselves as the precursors of a tumour. By the time a patient has a detectable solid tumour, cancer cells have escaped the initial immune response mechanisms. Here, we describe the development of a double binary zebrafish model that enables regulatory programming of the myeloid cells as they respond to oncogene-activated melanocytes to be explored, focussing on the initial phase when cells become the precursors of cancer. A hormone-inducible binary system allows for temporal control of expression of different Ras oncogenes (NRasQ61K, HRasG12V and KRasG12V) in melanocytes, leading to proliferation and changes in morphology of the melanocytes. This model was coupled to binary cell-specific biotagging models allowing in vivo biotinylation and subsequent isolation of macrophage or neutrophil nuclei for regulatory profiling of their active transcriptomes. Nuclear transcriptional profiling of neutrophils, performed as they respond to the earliest precursors of melanoma in vivo, revealed an intricate landscape of regulatory factors that may promote progression to melanoma, including Serpinb1l4, Fgf1, Fgf6, Cathepsin H, Galectin 1 and Galectin 3. The model presented here provides a powerful platform to study the myeloid response to the earliest precursors of melanoma. Summary: The authors present an innovative double binary zebrafish model for exploring regulatory mechanisms that govern the myeloid response at the onset of melanoma.