Activation of the Lck Tyrosine Kinase Targets Cell Surface T Cell Antigen Receptors for Lysosomal Degradation

AbstractThe mechanism by which TCR expression is regulated was explored by expressing a constitutively active form of the tyrosine kinase Lck (Lck505F) in T cells. Expression of Lck505F down-regulated TCR levels, an effect that was even more pronounced in CD45− T cells, in which the activity of this tyrosine kinase is further enhanced. Cells expressing Lck505F synthesized all TCR subunits, but lysosomal degradation of assembled receptors was enhanced. TCRs were rapidly internalized and degraded after removal of a tyrosine kinase inhibitor that had permitted cell surface expression. Finally, TCR levels on thymocytes were increased by an Lck inhibitor, and activation- but not phorbol ester–induced internalization of TCRs on Jurkat cells was prevented by inhibition or loss of Lck. These studies identify a regulated nonreceptor tyrosine kinase–mediated pathway for targeting cell surface receptors for lysosomal degradation

Exosome-delivered microRNAs promote IFN-α secretion by human plasmacytoid DCs via TLR7

The excessive production of type I IFNs is a hallmark and a main pathogenic mechanism of many autoimmune diseases, including systemic lupus erythematosus (SLE). In these pathologies, the sustained secretion of type I IFNs is dependent on the improper activation of plasmacytoid DCs (pDCs) by self-nucleic acids. However, the nature and origin of pDC-activating self-nucleic acids is still incompletely characterized. Here, we report that exosomes isolated from the plasma of SLE patients can activate the secretion of IFN-α by human blood pDCs in vitro. This activation requires endosomal acidification and is recapitulated by microRNAs isolated from exosomes, suggesting that exosome-delivered microRNAs act as self-ligands of innate single-stranded endosomal RNA sensors. By using synthetic microRNAs, we identified an IFN induction motif that is responsible for the TLR7-dependent activation, maturation, and survival of human pDCs. These findings identify exosome-delivered microRNAs as potentially novel TLR7 endogenous ligands able to induce pDC activation in SLE patients. Therefore, microRNAs may represent novel pathogenic mediators in the onset of autoimmune reactions and potential therapeutic targets in the treatment of type I IFN-mediated diseases

Vaccine Adjuvant MF59 Promotes Retention of Unprocessed Antigen in Lymph Node Macrophage Compartments and Follicular Dendritic Cells

Abstract Ag retention within lymph nodes (LNs) upon vaccination is critical for the development of adaptive immune responses, because it facilitates the encounter of the Ag with cognate lymphocytes. During a secondary exposure of the immune system to an Ag, immune complexes (ICs) that contain the unprocessed Ag are captured by subcapsular sinus macrophages and are transferred onto follicular dendritic cells, where they persist for weeks, facilitating Ag presentation to cognate memory B cells. The impact of adjuvants on Ag retention within the draining LNs is unknown. In this article, we provide the first evidence, to our knowledge, that the oil-in-water emulsion adjuvant MF59 localizes in subcapsular sinus and medullary macrophage compartments of mouse draining LNs, where it persists for at least 2 wk. In addition, we demonstrate that MF59 promotes accumulation of the unprocessed Ag within these LN compartments and facilitates the consequent deposition of the IC-trapped Ag onto activated follicular dendritic cells. These findings correlate with the ability of MF59 to boost germinal center generation and Ag-specific Ab titers. Our data suggest that the adjuvant effect of MF59 is, at least in part, due to an enhancement of IC-bound Ag retention within the LN and offer insights to improve the efficacy of new vaccine adjuvants

Serafino Zappacosta: An Enlightened Mentor and Educator.

With this article, the authors aim to honor the memory of Serafino Zappacosta, who had been their mentor during the early years of their career in science. The authors discuss how the combination of Serafino Zappacosta's extraordinary commitment to teaching and passion for science created a fostering educational environment that led to the creation of the "Ruggero Ceppellini Advanced School of Immunology." The review also illustrates how the research on the MHC and the inspirational scientific context in the Zappacosta's laboratory influenced the authors' early scientific interests, and subsequent professional work as immunologists

Src family kinases regulate interferon regulatory factor 1 K63 ubiquitination following activation by TLR7/8 vaccine adjuvant in human monocytes and B cells

Toll-like receptors (TLRs) play a key role in the activation of innate immune cells, in which their engagement leads to production of cytokines and co-stimulatory molecules. TLRs signaling requires recruitment of toll/IL-1R (TIR) domain-containing adaptors, such as MyD88 and/or TRIF, and leads to activation of several transcription factors, such as NF-κB, the AP1 complex, and various members of the interferon regulatory factor (IRF) family, which in turn results in triggering of several cellular functions associated with these receptors. A role for Src family kinases (SFKs) in this signaling pathway has also been established. Our work and that of others have shown that this type of kinases is activated following engagement of several TLRs, and that this event is essential for the initiation of specific downstream cellular response. In particular, we have previously demonstrated that activation of SFKs is required for balanced production of pro-inflammatory cytokines by monocyte-derived dendritic cells after stimulation with R848, an agonist of human TLRs 7/8. We also showed that TLR7/8 triggering leads to an increase in interferon regulatory factor 1 (IRF-1) protein levels and that this effect is abolished by inhibition of SFKs, suggesting a critical role of these kinases in IRF-1 regulation. In this study, we first confirmed the key role of SFKs in TLR7/8 signaling for cytokine production and accumulation of IRF-1 protein in monocytes and in B lymphocytes, two other type of antigen-presenting cells. Then, we demonstrate that TLR7 triggering leads to an increase of K63-linked ubiquitination of IRF-1, which is prevented by SFKs inhibition, suggesting a key role of these kinases in posttranslational regulation of IRF-1 in the immune cells. In order to understand the mechanism that links SFKs activation to IRF-1 K63-linked ubiquitination, we examined SFKs and IRF-1 possible interactors and proved that activation of SFKs is necessary for their interaction with TNFR-associated factor 6 (TRAF6) and promotes the recruitment of both cIAP2 and IRF-1 by TRAF6. Collectively, our data demonstrate that TLR7/8 engagement leads to the formation of a complex that allows the interaction of cIAP2 and IRF-1 resulting in IRF-1 K63-linked ubiquitination, and that active SFKs are required for this process

Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: role of HSP70/TLR2/NF-kB axis

Exosomes are a class of nanovesicles formed and released through the late endosomal compartment and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Here, we investigated whether genotoxic stress could promote the release of exosomes from multiple myeloma (MM) cells and studied the immunomodulatory properties they exert on NK cells, a major component of the antitumor immune response playing a key role in the immunosurveillance of MM. Our findings show that melphalan, a genotoxic agent used in MM therapy, significantly induces an increased exosome release from MM cells. MM cell-derived exosomes are capable of stimulating IFNg production, but not the cytotoxic activity of NK cells through a mechanism based on the activation of NF-kB pathway in a TLR2/ HSP70-dependent manner. Interestingly, HSP70 positive exosomes are primarily found in the bone marrow (BM) of MM patients suggesting that they might have a crucial immunomodulatory action in the tumor microenvironment. We also provide evidence that the CD56high NK cell subset is more responsive to exosome-induced IFNg production mediated by TLR2 engagement. All together, these findings suggest a novel mechanism of synergism between chemotherapy and antitumor innate immune responses based on the drug-promotion of nanovesicles exposing DAMPs for innate receptors

Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein

The immune response against hepatitis C virus (HCV) is rarely effective at clearing the virus, resulting in ∼170 million chronic HCV infections worldwide. Here we report that ligation of an HCV receptor (CD81) inhibits natural killer (NK) cells. Cross-linking of CD81 by the major envelope protein of HCV (HCV-E2) or anti-CD81 antibodies blocks NK cell activation, cytokine production, cytotoxic granule release, and proliferation. This inhibitory effect was observed using both activated and resting NK cells. Conversely, on NK-like T cell clones, including those expressing NK cell inhibitory receptors, CD81 ligation delivered a costimulatory signal. Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I. These results implicate HCV-E2–mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection

Design of a novel vaccine nanotechnology-based delivery system comprising CpGODN-protein conjugate anchored to liposomes

Although the well-known Toll like receptor 9 (TLR9) agonist CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity remain a concern. In an effort to circumvent these issues, different strategies have been employed to increase its stability, localise action and reduce dosage. These include conjugation of CpGODN with proteins or encapsulation/adsorption of CpGODN into/onto liposomes, and have resulted in enhanced immunopotency compared to co-administration of free CpGODN and antigen. Here, we designed a novel delivery system of CpGODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate the Group B Streptococcus (GBS) GBS67 protein antigen with the CpGODN TLR9 agonist. This treatment did not alter protein's ability to be recognised by specific antibodies or the CpGODN to function as a TLR9 agonist. Due to its negative charge, the protein conjugate readily electrostatically bound cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The novel cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following intramuscular immunisation, GBS67-CpGODN+L formed a vaccine depot at the injection site and induced a remarkable increase of functional immune responses against GBS compared to the simple co-administration of GBS67, CpGODN and liposomes. This work demonstrates that the conjugation of CpGODN to GBS67 in conjunction with adsorption on cationic liposomes, can promote co-delivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. Our findings highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms

DC-ATLAS: a systems biology resource to dissect receptor specific signal transduction in dendritic cells

BACKGROUND: The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs). RESULTS: Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules. CONCLUSIONS: The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies

Src Kinases Are Required for a Balanced Production of IL-12/IL-23 in Human Dendritic Cells Activated by Toll-Like Receptor Agonists

BACKGROUND: Pathogen recognition by dendritic cells (DC) is crucial for the initiation of both innate and adaptive immune responses. Activation of Toll-like Receptors (TLRs) by microbial molecular patterns leads to the maturation of DC, which present the antigen and activate T cells in secondary lymphoid tissues. Cytokine production by DC is critical for shaping the adaptive immune response by regulating T helper cell differentiation. It was previously shown by our group that Src kinases play a key role in cytokines production during TLR4 activation in human DC. PRINCIPAL FINDINGS: In this work we investigated the role of Src kinases during different TLRs triggering in human monocyte-derived DC (MoDC). We found that Src family kinases are important for a balanced production of inflammatory cytokines by human MoDC upon stimulation of TLR3 and 8 with their respective agonists. Disruption of this equilibrium through pharmacological inhibition of Src kinases alters the DC maturation pattern. In particular, while expression of IL-12 and other inflammatory cytokines depend on Src kinases, the induction of IL-23 and co-stimulatory molecules do not. Accordingly, DC treated with Src inhibitors are not compromised in their ability to induce CD4 T cell proliferation and to promote the Th17 subset survival but are less efficient in inducing Th1 differentiation. CONCLUSIONS: We suggest that the pharmacological modulation of DC maturation has the potential to shape the quality of the adaptive immune response and could be exploited for the treatment of inflammation-related diseases