Adaptive Immune Effector Research Articles

CCR5 deficiency/CCR5Δ32: resistant to HIV infection at the cost of curtailed CD4+ T cell memory responses.

Orchestrated trafficking and activation by pathogen-derived peptides define the ability of CD4+ T helper cells to contribute to an effective adaptive immunity. In this issue of The EMBO Journal, Martín-Leal etal show that the inflammatory chemokine receptor CCR5, well known for its role in cell migration and HIV infection, regulates ceramide synthesis and TCR nanoclustering to promote memory CD4+ T cell activation.

Lamprey VLRB participates in pathogen detection, VLRB/L-BLNK/L-NF-κB (B-like cells) signal transduction, and development

In jawed vertebrates, B cell receptors (BCR) are primary pathogen detectors that activate downstream signaling pathways to express adaptive immune effectors. In jawless vertebrates, the variable lymphocyte receptors (VLR) B positive lymphocytes can express and secrete specific VLRB molecules in an analogous manner to that of immunoglobulins by B cells in jawed vertebrates. Our study is the first to demonstrate the possibility of incubation of fertilized eggs and artificial breeding of Lampetra morii larvae throughout their life cycle under laboratory condition. We also found that VLRB, lamprey B-cell linker (L-BLNK), and lamprey nuclear factor-kappa B (L-NF-κB) play key roles in early larval development. Aeromonas hydrophila was found to be a lethal pathogen of L. morii larvae causing rapid infection at a concentration of 107 cfu/mL qRT-PCR results revealed that gene expression levels of VLRB, L-BLNK, and L-NF-κB were up-regulated significantly. Ten-day infection trials showed that VLRB, L-BLNK, and L-NF-κB are crucial for lamprey immune response. Furthermore, the expression levels of L-BLNK and L-NF-κB were down-regulated drastically both at mRNA and protein levels after bacterial infection than in the naive group of VLRB morphants. A similar expression pattern of VLRB and L-BLNK was found in L-NF-κB morphants post bacterial infection. The results were strikingly different in the other two morphants. The VLRB and L-NF-κB expression levels were found to be down-regulated at mRNA and protein levels by less than 30% and 45%, respectively, in L-BLNK morphants compared to those in the naive group. These results indicate that L-BLNK and L-NF-κB might participate in VLRB-mediated immune response. Additionally, in VLRB morphants, the mRNA expression levels of some genes, especially the ones expressed in VLRB+ lymphocytes but not in VLRA+ lymphocytes, were found to be affected. Therefore, these findings of B-like lymphocytes in lamprey offer key evidence with regard to the evolution of adaptive immunity.

Buruli ulcer: The Efficacy of Innate Immune Defense May Be a Key Determinant for the Outcome of Infection With Mycobacterium ulcerans.

Buruli ulcer (BU) is a neglected, tropical infectious disease of the skin and the subcutaneous tissue caused by Mycobacterium ulcerans. This pathogen has emerged as a new species from a common ancestor with Mycobacterium marinum by acquisition of the virulence plasmid pMUM. The plasmid encodes enzymes required for the synthesis of the macrolide toxin mycolactone, which has cytotoxic and immunosuppressive activities. In advanced BU lesions, extracellular clusters of M. ulcerans reside in necrotic subcutaneous tissue and are protected from infiltrating leukocytes by the cytotoxic activity of secreted mycolactone. Several lines of evidence indicate that elements of the innate immune system eliminate in many cases the initial inoculum before bacterial clusters can form and that therefore exposure to M. ulcerans leads only in a minority of individuals to the characteristic chronic necrotizing BU lesions. It is assumed that phagocytes play a key role in early host defense against M. ulcerans. Antibodies against bacterial surface structures seem to have less potential to enhance innate immunity than TH1 cell responses. Precise innate and adaptive immune effector mechanisms leading to protective immunity are however unclear, complicating the development of effective vaccines, the most desired solution to control BU. The tuberculosis vaccine Mycobacterium bovis Bacillus Calmette–Guérin (BCG) has limited short-term protective activity against BU. Whether this effect is due to the broad antigenic cross-reactivity between M. bovis and M. ulcerans or is at least partly mediated by a non-specific enhanced responsiveness of innate immune cells to secondary stimulation, recently described as “trained immunity” or “innate immune memory” is unknown but has major implications for vaccine design. Current vaccine research and development activities are focusing on recombinant BCG, subunit vaccines with selected M. ulcerans proteins, and the neutralization of mycolactone.

Minimal PD-1 expression in mouse and human NK cells under diverse conditions.

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Investigating the role of Quaking in antigen presentation by Dendritic Cells

Abstract Dendritic Cells (DCs) are considered the most potent antigen-presenting cells due to their superior capacity to cross-present exogenous antigens to CD8 T cells for effective adaptive immunity. They internalize foreign antigens by phagocytosis, receptor-mediated endocytosis or macropinocytosis, which are then processed in endo/lysosomal compartments and loaded on MHC class I. However, the molecular mechanisms regulating exogenous antigen uptake and cross-presentation by DCs remain unclear. We revealed a potential regulatory role of an RNA-binding protein, Quaking (QKI), in antigen presentation. Our previous studies in neural stem cells and microglia identified QKI as a novel regulator of phagosome maturation and endosome signaling via interaction with the transcription factor PPARβ/δ. Therefore, we hypothesize that QKI, together with PPARβ/δ enhances DC antigen uptake and cross-presentation via promoting phagosome and endolysosome signaling. We found QKI and PPARβ/δ to be significantly unregulated in human monocyte-derived DCs, and demonstrated that activating the QKI-PPARβ/δ complex in DCs upregulates QKI targets and significantly promotes DC phagocytosis activity, suggesting QKI cooperates with PPARβ/δ to enhance antigen uptake. The biological role of QKI in antigen cross-presentation and the effect of QKI enhancement on induction of CD8 T cell response by DC are currently under investigation. Understanding the role of QKI in DC may reveal how antigen presentation is regulated and provide new strategies to improve DC-based immunotherapy.

TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4+ T Cells to Treg

SUMMARYCD4+ T lymphocytes are crucial for controlling a range of innate and adaptive immune effectors. For CD8+ cytotoxic T lymphocyte (CTL) responses, CD4+ T cells can function as helpers (TH) to amplify magnitude and functionality or as regulatory cells (Treg) capable of profound inhibition. It is unclear what determines differentiation to these phenotypes and whether pathogens provoke alternate programs. We find that, depending on the size of initial dose, Listeria infection drives CD4+ T cells to act as TH or induces rapid polyclonal conversion to immunosuppressive Treg. Conversion to Treg depends on the TLR9 and IL-12 pathways elicited by CD8a+ dendritic cell (DC) sensing of danger-associated neutrophil self-DNA. These findings resolve long-standing questions regarding the conditional requirement for TH amongst pathogens and reveal a remarkable degree of plasticity in the function of CD4+ T cells, which can be quickly converted to Treg in vivo by infection-mediated immune modulation.

Molecular regulation of the plasma membrane-proximal cellular steps involved in NK cell cytolytic function.

Natural killer (NK) cells, cytolytic lymphocytes of the innate immune system, play a crucial role in the immune response against infection and cancer. NK cells kill target cells through exocytosis of lytic granules that contain cytotoxic proteins, such as perforin and granzymes. Formation of a functional immune synapse, i.e. the interface between the NK cell and its target cell enhances lysis through accumulation of polymerized F-actin at the NK cell synapse, leading to convergence of lytic granules to the microtubule organizing center (MTOC) and its subsequent polarization along microtubules to deliver the lytic granules to the synapse. In this review, we focus on the molecular mechanisms regulating the cellular processes that occur after the lytic granules are delivered to the cytotoxic synapse. We outline how - once near the synapse - the granules traverse the clearings created by F-actin remodeling to dock, tether and fuse with the plasma membrane in order to secrete their lytic content into the synaptic cleft through exocytosis. Further emphasis is given to the role of Ca2+ mobilization during degranulation and, whenever applicable, we compare these mechanisms in NK cells and cytotoxic T lymphocytes (CTLs) as adaptive immune system effectors.

The differential account of white blood cell as an image of immune system from flying fox (Pteropus spp) as a reservoir host

Flying fox (Pteropus spp) has been reported as a host reservoir of several microorganisms. Reservoir host is a species that serves primarily as a maintenance host for a pathogen related to the immune system. The purpose of this research is to describe the white blood cells differential account of Flying-foxes as a picture of the immune system and bioindicator of environmental changes. The Bloods are collected from The two adult Flying fox bats in December 2018. They were bought from a hunter in Wera Village, Bima district, Sumbawa Island. The Blood samples were collected from the axillaries vein of the bat for blood smears. The blood smears were Giemsa stained then examined for The white blood cells differential counts. The differential white blood cell (WBC) counts (%) of blood smear of Flying foxes are Eosinophils (2.25±1.71%), Neutrophils (14.00±2.45%), Basophils (13.75±4.11%), Monocytes (29.75±4.99%), and a Lymphocytes (40.25±9.03%). The highest percentage of Lymphocytes describe of the effectors of adaptive immunity and the highest percentage of Monocytes describe of the effectors of innate immunity. The lymphocytes and monocytes have an important role in the immune system and can be bio-indicator of environmental changes.

Abstract I02: Targeting Innate immunity in pancreatic carcinoma

Abstract Pancreatic carcinoma has proven largely resistant to immunotherapies as strategies directly targeting T cells or their receptors have failed in early clinical trials. Basic research has shown that effector T-cell infiltration and phenotype in pancreatic cancer is controlled by diverse innate immune elements including dendritic cells, macrophages and other myeloid cells, and subsets of innate-like T cells. However, the phenotype of macrophages and myeloid cells infiltrating pancreatic carcinoma is, for the most part, highly suppressive of effective adaptive antitumor immunity. Therefore, rather than directly targeting T cells in isolation, immunotherapeutic strategies addressing toleorogenic innate immune cell populations hold greater promise. New potential avenues for immunotherapy include activating or blockade of select innate immune receptors on tumor-associated macrophages to reprogram their suppressive phenotype, targeting the microbiome to modulate the intratumoral inflammatory environment toward a more favorable phenotype, or deleting select subsets of tolerogenic T cells such as delta1 cells. These type of approaches have the potential to enable efficacy for more traditional immunotherapies that target checkpoint or costimulatory receptors, and these strategies can also potentiate the effects of systemic chemotherapy or radiation therapy. Citation Format: George Miller. Targeting Innate immunity in pancreatic carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I02.

An Atlas of Clinically-Distinct Tumor Cellular Ecosystems in Diffuse Large B Cell Lymphoma

Background: Diffuse large B cell lymphoma (DLBCL) exhibits significant clinical and biological heterogeneity, in part due to cell-of-origin subtypes, somatic alterations, and diverse stromal constituents within the tumor microenvironment (TME). Several immunologically-active lymphoma therapies are known to rely on innate and adaptive anti-tumor responses occurring within this dynamic TME, including agents that are approved (e.g., rituximab, lenalidomide, CART19, ibrutinib) or emerging (e.g., anti-CD47, checkpoint inhibitors). We hypothesized that a large-scale characterization of the cellular heterogeneity in DLBCL might reveal previously unknown biological variation in the TME linked to tumor subtypes and genotypes, therapeutic responses and clinical outcomes, with implications for future personalization of immunotherapy. Methods: Using a combination of lymphoma single-cell RNA sequencing (scRNA-seq) and bulk tumor transcriptome deconvolution (CIBERSORTx; Newman et al., Nat Biotech, 2019), we developed a new machine learning framework for identifying cellular states and ecosystems that reflect fundamental TME subtypes and distinctions in tumor biology (Fig. 1). Specifically, using CIBERSORTx, we purified the transcriptomes of B cells and 12 different TME cell types, including immune and stromal subsets, from 1,279 DLBCL tumor biopsies profiled in 3 prior studies (Reddy et al., Cell 2017; Schmitz et al., NEJM 2018; Chapuy et al., Nat Med 2018). Then, we defined distinct transcriptional states for each of the 13 cell types, which we validated at single-cell resolution, using a combination of two scRNA-seq techniques (Smart-Seq2 and 10x Chromium 5' GEP, BCR and TCR) to profile primary DLBCL, FL, and human tonsils, as well as leveraging multiple scRNA-seq datasets from previous studies. We identified robust co-associations between cell states that form tumor cellular ecosystems, which we validated in independent datasets of bulk DLBCL tumor gene expression profiles. Finally, we related TME ecosystems to defined tumor subtypes, including genotype classes, and to clinical outcomes. Results: By systematically characterizing the landscape of cellular heterogeneity in nearly 1,300 DLBCL tumors, we defined an atlas of 49 distinct transcriptional states across 13 major cell types. These novel cell states spanned diverse innate and adaptive immune effector cells of the lymphoid and myeloid lineages, as well as tumor-associated fibroblasts. Remarkably, 94% of these states (46 of 49) could be validated in a compendium of ~200,000 single-cell transcriptomes derived from lymphomas, healthy control tonsils, and other tissue types. Moreover, single cells from DLBCL, FL and tonsils best mirrored these newly discovered cell states. We next characterized the biology and potential clinical utility of each cell state. We observed clear distinctions in the transcriptional programs of immune and stromal elements between germinal center and activated B cell DLBCL, as well as between known mutational subtypes. Importantly, many cell states reflected novel phenotypic groupings, and the majority were significantly associated with overall survival (P<0.05). These findings were highly concordant both within and across 3 independent DLBCL cohorts. By identifying groups of DLBCL patients with similar communities of cellular states, we defined cohesive cellular ecosystems that collectively capture the landscape of transcriptional heterogeneity in DLBCL tumors. Patients whose tumors were assigned to these ecosystems exhibited striking variation in overall survival. Importantly, the ecosystems defined distinct subgroups that could not be fully recapitulated by known transcriptional and genetic subtypes. Moreover, several TME classes showed significant enrichments in canonical or novel tumor genotypes, suggesting an evolutionary interplay between the tumor and host microenvironment. Conclusion: We describe a novel computational framework to digitally dissect the DLBCL TME and an atlas of novel states for diverse cell types in these tumors. We show how cellular states form cohesive tumor ecosystems, which exhibit distinct clinical outcomes and novel somatic alterations. These results expand our understanding of cellular heterogeneity in DLBCL, with implications for the development of individualized immunotherapies. Disclosures Kurtz: Roche: Consultancy. Advani:Kura: Research Funding; Merck: Research Funding; Millennium: Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cell Medica, Ltd: Consultancy; Kyowa Kirin Pharmaceutical Developments, Inc.: Consultancy; Stanford University: Employment, Equity Ownership; Janssen: Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Research Funding; Infinity Pharma: Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Celmed: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forty-Seven: Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc./Kite Pharma, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agensys: Research Funding. Diehn:Roche: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; BioNTech: Consultancy; Quanticell: Consultancy. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

Gene Expression-Based Identification of Antigen-Responsive CD8+ T Cells on a Single-Cell Level.

CD8+ T cells are important effectors of adaptive immunity against pathogens, tumors, and self antigens. Here, we asked how human cognate antigen-responsive CD8+ T cells and their receptors could be identified in unselected single-cell gene expression data. Single-cell RNA sequencing and qPCR of dye-labeled antigen-specific cells identified large gene sets that were congruently up- or downregulated in virus-responsive CD8+ T cells under different antigen presentation conditions. Combined expression of TNFRSF9, XCL1, XCL2, and CRTAM was the most distinct marker of virus-responsive cells on a single-cell level. Using transcriptomic data, we developed a machine learning-based classifier that provides sensitive and specific detection of virus-responsive CD8+ T cells from unselected populations. Gene response profiles of CD8+ T cells specific for the autoantigen islet-specific glucose-6-phosphatase catalytic subunit-related protein differed markedly from virus-specific cells. These findings provide single-cell gene expression parameters for comprehensive identification of rare antigen-responsive cells and T cell receptors.

Interferon-λ modulates dendritic cells to facilitate T cell immunity during infection with influenza A virus.

Type III interferon (IFN-λ) is important for innate immune protection at mucosal surfaces and has therapeutic benefit against influenza A virus (IAV) infection. However, the mechanisms by which IFN-λ programs adaptive immune protection against IAV are undefined. Here we found that IFN-λ signaling in dendritic cell (DC) populations was critical for the development of protective IAV-specific CD8+ T cell responses. Mice lacking the IFN-λ receptor (Ifnlr1–/–) had blunted CD8+ T cell responses relative to wildtype and exhibited reduced survival after heterosubtypic IAV re-challenge. Analysis of DCs revealed IFN-λ signaling directed the migration and function of CD103+ DCs for development of optimal anti-viral CD8+ T cell responses, and bioinformatic analyses identified IFN-λ regulation of a DC IL-10 immunoregulatory network. Thus, IFN-λ serves a critical role in bridging innate and adaptive immunity from lung mucosa to lymph nodes to program DCs to direct effective T cell immunity against IAV.

The Avidity Game: Selecting Natural-Born Killers

Natural killer (NK) cells display some features equivalent to those of adaptive immune effectors, but the molecular processes underlying these adaptive-like characteristics are just beginning to be defined. In this issue of Immunity, Adams etal. (2019) and Grassmann etal. (2019) report that avidity selection for Ly49H governs the expansion, differentiation, and function of NK cells after cytomegalovirus infection.

The Chemoattractant Receptor Ebi2 Drives Intranodal Naive CD4+ T Cell Peripheralization to Promote Effective Adaptive Immunity

Lymph nodes (LNs) play critical roles in adaptive immunity by concentrating in one location the antigens, antigen-presenting cells, and antigen-responsive lymphocytes involved in such responses. Recent studies have revealed nonrandom localization of innate and adaptive immune cells within these organs, suggesting that microanatomical positioning optimizes responses involving sparse cooperating cells. Here, we report that the peripheral localization of LN cDC2 dendritic cells specialized for MHC-II antigen presentation is matched by a similarly biased paracortical distribution of CD4+ Tcells directed by the chemoattractant receptor Ebi2. In the absence of Ebi2, CD4+ Tcells lose their location bias and are delayed in antigen recognition, proliferative expansion, differentiation, direct effector activity, and provision of help for CD8+ Tcell-mediated memory responses, limiting host defense and vaccine responses. These findings demonstrate evolutionary selection for distinct niches within the LN that promote cellular responses, emphasizing the critical link between fine-grained tissue organization and host defense.

Sources of Type I Interferons in Infectious Immunity: Plasmacytoid Dendritic Cells Not Always in the Driver's Seat.

Type I Interferons (IFNs) are hallmark cytokines produced in immune responses to all classes of pathogens. Type I IFNs can influence dendritic cell (DC) activation, maturation, migration, and survival, but also directly enhance natural killer (NK) and T/B cell activity, thus orchestrating various innate and adaptive immune effector functions. Therefore, type I IFNs have long been considered essential in the host defense against virus infections. More recently, it has become clear that depending on the type of virus and the course of infection, production of type I IFN can also lead to immunopathology or immunosuppression. Similarly, in bacterial infections type I IFN production is often associated with detrimental effects for the host. Although most cells in the body are thought to be able to produce type I IFN, plasmacytoid DCs (pDCs) have been termed the natural “IFN producing cells” due to their unique molecular adaptations to nucleic acid sensing and ability to produce high amounts of type I IFN. Findings from mouse reporter strains and depletion experiments in in vivo infection models have brought new insights and established that the role of pDCs in type I IFN production in vivo is less important than assumed. Production of type I IFN, especially the early synthesized IFNβ, is rather realized by a variety of cell types and cannot be mainly attributed to pDCs. Indeed, the cell populations responsible for type I IFN production vary with the type of pathogen, its tissue tropism, and the route of infection. In this review, we summarize recent findings from in vivo models on the cellular source of type I IFN in different infectious settings, ranging from virus, bacteria, and fungi to eukaryotic parasites. The implications from these findings for the development of new vaccination and therapeutic designs targeting the respectively defined cell types are discussed.

Different Faces for Different Places: Heterogeneity of Neutrophil Phenotype and Function.

As the most abundant leukocytes in the circulation, neutrophils are committed to innate and adaptive immune effector function to protect the human body. They are capable of killing intruding microbes through various ways including phagocytosis, release of granules, and formation of extracellular traps. Recent research has revealed that neutrophils are heterogeneous in phenotype and function and can display outstanding plasticity in both homeostatic and disease states. The great flexibility and elasticity arm neutrophils with important regulatory and controlling functions in various disease states such as autoimmunity and inflammation as well as cancer. Hence, this review will focus on recent literature describing neutrophils' variable and diverse phenotypes and functions in different contexts.

T Lymphocytes as Measurable Targets of Protection and Vaccination Against Viral Disorders.

Continuous epidemiological surveillance of existing and emerging viruses and their associated disorders is gaining importance in light of their abilities to cause unpredictable outbreaks as a result of increased travel and vaccination choices by steadily growing and aging populations. Close surveillance of outbreaks and herd immunity are also at the forefront, even in industrialized countries, where previously eradicated viruses are now at risk of re-emergence due to instances of strain recombination, contractions in viral vector geographies, and from their potential use as agents of bioterrorism. There is a great need for the rational design of current and future vaccines targeting viruses, with a strong focus on vaccine targeting of adaptive immune effector memory T cells as the gold standard of immunity conferring long-lived protection against a wide variety of pathogens and malignancies. Here, we review viruses that have historically caused large outbreaks and severe lethal disorders, including respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic fevers. To observe trends in vaccinology against these viral disorders, we describe viral genetic, replication, transmission, and tropism, host-immune evasion strategies, and the epidemiology and health risks of their associated syndromes. We focus on immunity generated against both natural infection and vaccination, where a steady shift in conferred vaccination immunogenicity is observed from quantifying activated and proliferating, long-lived effector memory T cell subsets, as the prominent biomarkers of long-term immunity against viruses and their associated disorders causing high morbidity and mortality rates.

Complement C3 Plays a Key Role in Inducing Humoral and Cellular Immune Responses to Influenza Virus Strain-Specific Hemagglutinin-Based or Cross-Protective M2 Extracellular Domain-Based Vaccination.

The complement pathway is involved in eliminating antigen immune complexes. However, the role of the C3 complement system remains largely unknown in influenza virus M2 extracellular (M2e) domain or hemagglutinin (HA) vaccine-mediated protection after vaccination. Using a C3 knockout (C3 KO) mouse model, we found that complement protein C3 was required for effective induction of immune responses to vaccination with M2e-based or HA-based vaccines, which include isotype class-switched antibodies and effector CD4 and CD8 T cell responses. C3 KO mice after active immunization with cross-protective nonneutralizing M2e-based vaccine were not protected against influenza virus, although low levels of M2e-specific antibodies were protective after passive coadministration with virus in wild-type mice. In contrast, C3 KO mice that were immunized with strain-specific neutralizing HA-based vaccine were protected against homologous virus challenge despite lower levels of HA antibody responses. C3 KO mice showed impaired maintenance of innate immune cells and a defect in innate immune responses upon exposure to antigens. The findings in this study suggest that C3 is required for effective induction of humoral and cellular adaptive immune responses as well as protective immunity after nonneutralizing influenza M2e vaccination.IMPORTANCE Complement is the well-known innate immune defense system involved in the opsonization and lysis of pathogens but is less studied in establishing adaptive immunity after vaccination. Influenza virus HA-based vaccination confers protection via strain-specific neutralizing antibodies, whereas M2e-based vaccination induces a broad spectrum of protection by immunity against the conserved M2e epitopes. This study revealed the critical roles of C3 complement in inducing humoral and cellular immune responses after immunization with M2e or HA vaccines. C3 was found to be required for protection by M2e-based but not by HA-based active vaccination as well as for maintaining innate antigen-presenting cells. Findings in this study have insight into better understanding the roles of C3 complement in inducing effective innate and adaptive immunity as well as in conferring protection by cross-protective conserved M2e vaccination.

TLR9-mediated activation of dendritic cells by CD32 targeting for the generation of highly immunostimulatory vaccines

ABSTRACTThe rational for designing dendritic cell (DC)-targeted immunotherapies is their central role in orchestrating immunity. Most studies addressing antigen-targeting to DCs for eliciting T cell responses have employed ex-vivo matured DCs derived from monocytes or myeloid DCs isolated from peripheral blood. More recently, also plasmacytoid DCs (pDCs) emerged as attractive targets that can be readily isolated and activated ex vivo. pDCs are known as key effectors of innate and adaptive immunity due to their exquisite ability to produce large amounts of type-1 interferons upon signaling via TLR7 or TLR9 intracellular receptor for viral RNA or bacterial DNA, respectively. In this study, we describe and characterize the immune modulating and targeting module of a composite human specific vaccine platform for active immunotherapy. This module, called warhead (WH), is composed of a single-chain variable fragment (scFv) and CpG-C type oligonucleotides (ODNs) that are covalently coupled. The scFv mediates specific binding to FcγRII/CD32 on APCs and internalization of the ODNs which stimulate TLR9-expressing B cells and pDCs. Furthermore, the scFv in the WH is extended with a five-time heptad repeat (EVSALEK) alpha helix which allows for a coiled-coil complex formation with any immunogen also extended with another five-time heptad (KVSALKE) repeat. WH elicits fast and robust pDC activation as evidenced by the release of interferon-α, TNF-α and IL-6. The WH thus takes advantage of the key features of human pDCs for immunostimulation and can be a versatile tool for antigen-specific vaccination with a variety of proteins or peptides.

Messing with the Sentinels-The Interaction of Staphylococcus aureus with Dendritic Cells.

Staphylococcus aureus (S. aureus) is a dangerous pathogen as well as a frequent colonizer, threatening human health worldwide. Protection against S. aureus infection is challenging, as the bacteria have sophisticated strategies to escape the host immune response. To maintain equilibrium with S. aureus, both innate and adaptive immune effector mechanisms are required. Dendritic cells (DCs) are critical players at the interface between the two arms of the immune system, indispensable for inducing specific T cell responses. In this review, we highlight the importance of DCs in mounting innate as well as adaptive immune responses against S. aureus with emphasis on their role in S. aureus-induced respiratory diseases. We also review what is known about mechanisms that S. aureus has adopted to evade DCs or manipulate these cells to its advantage.