Testing S. sonnei GMMA with and without Aluminium Salt-Based Adjuvants in Animal Models.

Shigellosis is one of the leading causes of diarrheal disease in low- and middle-income countries, particularly in young children, and is more often associated with antimicrobial resistance. Therefore, a preventive vaccine against shigellosis is an urgent medical need. We have proposed Generalised Modules for Membrane Antigens (GMMA) as an innovative delivery system for Shigella sonnei O-antigen, and an Alhydrogel formulation (1790GAHB) has been extensively tested in preclinical and clinical studies. Alhydrogel has been used as an adsorbent agent with the main purpose of reducing potential GMMA systemic reactogenicity. However, the immunogenicity and systemic reactogenicity of this GMMA-based vaccine formulated with or without Alhydrogel have never been compared. In this work, we investigated the potential adjuvant effect of aluminium salt-based adjuvants (Alhydrogel and AS37) on S. sonnei GMMA immunogenicity in mice and rabbits, and we found that S. sonnei GMMA alone resulted to be strongly immunogenic. The addition of neither Alhydrogel nor AS37 improved the magnitude or the functionality of vaccine-elicited antibodies. Interestingly, rabbits injected with either S. sonnei GMMA adsorbed on Alhydrogel or S. sonnei GMMA alone showed a limited and transient body temperature increase, returning to baseline values within 24 h after each vaccination. Overall, immunisation with unadsorbed GMMA did not raise any concern for animal health. We believe that these data support the clinical testing of GMMA formulated without Alhydrogel, which would allow for further simplification of GMMA-based vaccine manufacturing.

The overlooked bacterial pandemic.

The COVID-19 pandemic had a significant economic and health impact worldwide. It also reinforced the misperception that only viruses can pose a threat to human existence, overlooking that bacteria (e.g., plague and cholera) have severely haunted and shaped the course of human civilization. While the world is preparing for the next viral pandemic, it is again overlooking a silent one: antimicrobial resistance (AMR). This review proposes to show the impact of bacterial infections on civilization to remind the pandemic potential. The work will also discuss a few examples of how bacteria can mutate risking global spread and devastating outcomes, the effect on the global burden, and the prophylactic and therapeutic measures. Indeed, AMR is dramatically increasing and if the trend is not reversed, it has the potential to quickly turn into the most important health problem worldwide.

Enhanced Systemic Humoral Immune Response Induced in Mice by Generalized Modules for Membrane Antigens (GMMA) Is Associated with Affinity Maturation and Isotype Switching.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria that can be used to design affordable subunit vaccines. GMMA have been observed to induce a potent humoral immune response in preclinical and clinical studies. In addition, in preclinical studies, it has been found that GMMA can be exploited as optimal antigen carriers for both protein and saccharide antigens, as they are able to promote the enhancement of the antigen-specific humoral immune response when the antigen is overexpressed or chemically conjugated to GMMA. Here we investigated the mechanism of this GMMA carrier effect by immunizing mice and using factor H binding protein and GMMA of Neisseria meningitidis B as an antigen-GMMA model. We confirmed that the antigen displayed on the GMMA surface increased the antigen-specific IgG production and, above all, the antibody functionality measured by the serum bactericidal activity. We found that the enhancement of the bactericidal capacity induced by GMMA carrying the antigen on the surface was associated with the increase in antibody affinity to the antigen, and with the switching toward IgG subclasses with more bactericidal potential. Thus, we conclude that the potent carrier effect of GMMA is due to their ability to promote a better quality of humoral immunity.

Challenges in development of vaccines directed toward antimicrobial resistant bacterial species.

Antimicrobial resistance (AMR) is considered by WHO one of the top ten public health threats. New control strategies involving concerted actions of both public and private sectors need to be developed. Vaccines play a major role in controlling the spread of AMR pathogens by decreasing transmission and limiting the use of antibiotics, reducing at the end the selective pressure for the emergence of new resistant strains. In this review, by using as example some of the most serious AMR pathogens, we highlighted the major hurdles from a research and development point of view. New approaches to better understand the immunological mechanisms of response to both natural infections and vaccines that aimed to identify correlates of protection, together with the application of new technologies for vaccine design and delivery are discussed as potential solutions.

Exploring the Role of GMMA Components in the Immunogenicity of a 4-Valent Vaccine against Shigella.

Shigellosis is the leading cause of diarrheal disease, especially in children of low- and middle-income countries, and is often associated with anti-microbial resistance. Currently, there are no licensed vaccines widely available against Shigella, but several candidates based on the O-antigen (OAg) portion of lipopolysaccharides are in development. We have proposed Generalized Modules for Membrane Antigens (GMMA) as an innovative delivery system for OAg, and a quadrivalent vaccine candidate containing GMMA from S. sonnei and three prevalent S. flexneri serotypes (1b, 2a and 3a) is moving to a phase II clinical trial, with the aim to elicit broad protection against Shigella. GMMA are able to induce anti-OAg-specific functional IgG responses in animal models and healthy adults. We have previously demonstrated that antibodies against protein antigens are also generated upon immunization with S. sonnei GMMA. In this work, we show that a quadrivalent Shigella GMMA-based vaccine is able to promote a humoral response against OAg and proteins of all GMMA types contained in the investigational vaccine. Proteins contained in GMMA provide T cell help as GMMA elicit a stronger anti-OAg IgG response in wild type than in T cell-deficient mice. Additionally, we observed that only the trigger of Toll-like Receptor (TLR) 4 and not of TLR2 contributed to GMMA immunogenicity. In conclusion, when tested in mice, GMMA of a quadrivalent Shigella vaccine candidate combine both adjuvant and carrier activities which allow an increase in the low immunogenic properties of carbohydrate antigens.

Antigen presentation by Follicular Dendritic cells to cognate B cells is pivotal for Generalised Modules for Membrane Antigens (GMMA) immunogenicity.

GMMA has been proposed as a potent technology platform for the design of safe, effective and affordable vaccines. As GMMA are vesicles blebbing out of the outer membrane of Gram-negative bacteria, they contain lipopolysaccharides, lipoproteins and peptidoglycans that stimulate immune cells via Toll-like Receptors 4 (TLR4) or TLR2. Being basically nanoparticles, GMMA can be efficiently captured by Follicular Dendritic Cells (FDC) for antigen presentation to cognate B cells. GMMA have shown to be highly immunogenic in preclinical and clinical studies and the engagement of TLR4 and TLR2 or antigen presentation by FDC may have a prominent role in GMMA immunogenicity, which is well worth investigating. By using GMMA derived from Shigella sonnei and Salmonella Typhimurium, we show for the first time that the antigen presentation by FDC to cognate B cells plays a major role in the induction of an effective humoral immune response upon immunization with GMMA by using both models. The engagement of TLR4 is critical to elicit an optimal antibody production, but its effect on antibody functionality is dependent on GMMA type and is dispensable when immunizing with Alum adjuvant, whereas TLR2 does not have any role for GMMA immunogenicity. Our findings represent a substantial advancement of the knowledge on GMMA mode of action and shed a light on novel perspectives for the design of safer and more effective GMMA-based vaccines. ONE SENTENCE SUMMARY: The study demonstrated that the antigen presentation by FDC to cognate B cells plays a major role for GMMA immunogenicity.

GMMA as a 'plug and play' technology to tackle infectious disease to improve global health: context and perspectives for the future.

INTRODUCTION: Generalized-Modules-for-Membrane-Antigens (GMMA) is a technology platform developed to design outer membrane vesicle (OMV)-based vaccines. GMMA are basically OMVs derived from a bacterial strain specifically engineered to obtain a fit-for-purpose and affordable vaccine by potentiating, or deleting, expression of specific genes. OMVs can be used as a carrier for antigens by inducing their expression on them, with the aim to improve antigen immunogenicity and design multivalent combination vaccines. AREAS COVERED: We expanded this finding to show that the chemical conjugation of different proteic and/or polysaccharidic antigens, to GMMA, is a methodology complementary to the genetic manipulation to obtain highly effective combination vaccines. Here we discuss our findings with a specific focus on the impact that GMMA technology can have on global health, as this technology platform is particularly suited to support the development of affordable vaccines for low-income countries. EXPERT OPINION: We believe that it is critical to elucidate the mode of action of GMMA immunogenicity and have provided a summarized description of the immunological questions to be addressed in the near future. The improved knowledge of GMMA might lead to designing more effective and safer GMMA-based vaccines to tackle the most serious vaccine-preventable diseases.

Generalized Modules for Membrane Antigens as Carrier for Polysaccharides: Impact of Sugar Length, Density, and Attachment Site on the Immune Response Elicited in Animal Models.

Nanoparticle systems are being explored for the display of carbohydrate antigens, characterized by multimeric presentation of glycan epitopes and special chemico-physical properties of nano-sized particles. Among them, outer membrane vesicles (OMVs) are receiving great attention, combining antigen presentation with the immunopotentiator effect of the Toll-like receptor agonists naturally present on these systems. In this context, we are testing Generalized Modules for Membrane Antigens (GMMA), OMVs naturally released from Gram-negative bacteria mutated to increase blebbing, as carrier for polysaccharides. Here, we investigated the impact of saccharide length, density, and attachment site on the immune response elicited by GMMA in animal models, using a variety of structurally diverse polysaccharides from different pathogens (i.e., Neisseria meningitidis serogroup A and C, Haemophilus influenzae type b, and streptococcus Group A Carbohydrate and Salmonella Typhi Vi). Anti-polysaccharide immune response was not affected by the number of saccharides per GMMA particle. However, lower saccharide loading can better preserve the immunogenicity of GMMA as antigen. In contrast, saccharide length needs to be optimized for each specific antigen. Interestingly, GMMA conjugates induced strong functional immune response even when the polysaccharides were linked to sugars on GMMA. We also verified that GMMA conjugates elicit a T-dependent humoral immune response to polysaccharides that is strictly dependent on the nature of the polysaccharide. The results obtained are important to design novel glycoconjugate vaccines using GMMA as carrier and support the development of multicomponent glycoconjugate vaccines where GMMA can play the dual role of carrier and antigen. In addition, this work provides significant insights into the mechanism of action of glycoconjugates.

GMMA Is a Versatile Platform to Design Effective Multivalent Combination Vaccines.

Technology platforms are an important strategy to facilitate the design, development and implementation of vaccines to combat high-burden diseases that are still a threat for human populations, especially in low- and middle-income countries, and to address the increasing number and global distribution of pathogens resistant to antimicrobial drugs. Generalized Modules for Membrane Antigens (GMMA), outer membrane vesicles derived from engineered Gram-negative bacteria, represent an attractive technology to design affordable vaccines. Here, we show that GMMA, decorated with heterologous polysaccharide or protein antigens, leads to a strong and effective antigen-specific humoral immune response in mice. Importantly, GMMA promote enhanced immunogenicity compared to traditional formulations (e.g., recombinant proteins and glycoconjugate vaccines), without negative impact to the anti-GMMA immune response. Our findings support the use of GMMA as a "plug and play" technology for the development of effective combination vaccines targeting different bugs at the same time.

Toll-like receptor 4 signaling in hematopoietic-lineage cells contributes to the enhanced activity of the human vaccine adjuvant AS01.

The 3-O-desacyl-4'-monophosphoryl lipid A (MPL) activates immunity through Toll-like receptor 4 (TLR4) signaling. The Adjuvant System AS01 contains MPL and is used in the candidate malaria vaccine and the licensed zoster vaccine. Recent studies reported that AS01 adjuvant activity depends on a transient inflammation at the site of vaccination, but the role of stromal or structural cells in the adjuvant effect is unknown. We investigated this question in mouse models by assessing the role of TLR4 on hematopoietic versus resident structural cells during immunization with AS01-adjuvanted vaccines. We first established that TLR4-deficient animals had a reduced immune response to an AS01-adjuvanted vaccine. Using bone marrow chimera, we consistently found that Tlr4 expression in radio-sensitive cells, i.e., hematopoietic cells, was required for an optimal adjuvant effect on antibody and T-cell responses. At day 1 after injection, the pro-inflammatory reaction at the site of injection was strongly dependent on TLR4 signaling in hematopoietic cells. Similarly, activation of dendritic cells in muscle-draining lymph nodes was strictly associated with the radio-sensitive cells expressing Tlr4. Altogether, these data suggest that MPL-mediated TLR4-signaling in hematopoietic cells is critical in the mode of action of AS01.

Co-administration of GM-CSF expressing RNA is a powerful tool to enhance potency of SAM-based vaccines.

Self-amplifying mRNAs (SAM)-based vaccines have been shown to induce a robust immune response in various animal species against both viral and bacterial pathogens. Due to their synthetic nature and to the versatility of the manufacturing process, SAM technology may represent an attractive solution for rapidly producing novel vaccines, which is particularly critical in case of pandemic infections or diseases mediated by newly emerging pathogens. Recent published data support the hypothesis that Antigen Presenting Cells (APCs) are responsible for CD8+ T-cell priming after SAM vaccination, suggesting cross-priming as the key mechanism for antigen presentation by SAM vaccines. In our study we investigated the possibility to enhance the immune response induced in mice by a single immunization with SAM by increasing the recruitment of APCs at the site of injection. To enhance SAM immunogenicity, we selected murine granulocyte-macrophage colony-stimulating factor (GM-CSF) as a model chemoattractant for APCs, and developed a SAM-GM-CSF vector. We evaluated whether the use of SAM-GM-CSF in combination with a SAM construct encoding the Influenza A virus nucleoprotein (NP) would lead to an increase of APC recruitment and NP-specific immune response. We indeed observed that the administration of SAM-GM-CSF enhances the recruitment of APCs at the injection site. Consistently with our hypothesis, co-administration of SAM-GM-CSF with SAM-NP significantly improved the magnitude of NP-specific CD8+ T-cell response both in terms of frequency of cytotoxic antigen-specific CD8+ T-cells and their functional activity in vivo. Furthermore, co-immunization with SAM-GM-CSF and SAM-NP provided an increase in protection against a lethal challenge with influenza virus. In conclusion, we demonstrated that increased recruitment of APCs at the site of injection is associated with an enhanced effectiveness of SAM vaccination and might be a powerful tool to potentiate the efficacy of RNA vaccination.

Alum/Toll-Like Receptor 7 Adjuvant Enhances the Expansion of Memory B Cell Compartment Within the Draining Lymph Node.

Vaccination is one of the most cost-effective health interventions and, with the exception of water sanitization, no other action has had such a major effect in mortality reduction. Combined with other approaches, such as clean water, better hygiene, and health education, vaccination contributed to prevent millions of cases of deaths among children under 5 years of age. New or improved vaccines are needed to fight some vaccine-preventable diseases that are still a threat for the public health globally, as reported also in the Global Vaccine Action Plan (GVAP) endorsed by the World Health Assembly in 2012. Adjuvants are substances that enhance the effectiveness of vaccination, but despite their critical role for the development of novel vaccines, very few of them are approved for use in humans. Aluminum hydroxide (Alum) is the most common adjuvant used in vaccines administered in millions of doses around the world to prevent several dangerous diseases. The development of an improved version of Alum can help to design and produce new or better vaccines. Alum/toll-like receptor (TLR)7 is a novel Alum-based adjuvant, currently in phase I clinical development, formed by the attachment of a benzonaphthyridine compound, TLR7 agonist, to Alum. In preclinical studies, Alum/TLR7 showed a superior adjuvant capacity, compared to Alum, in several disease models, such as meningococcal meningitis, anthrax, staphylococcus infections. None of these studies reported the effect of Alum/TLR7 on the generation of the B cell memory compartment, despite this is a critical aspect to achieve a better immunization. In this study, we show, for the first time, that, compared to Alum, Alum/TLR7 enhances the expansion of the memory B cell compartment within the draining lymph node (LN) as result of intranodal sustained proliferation of antigen-engaged B cells and/or accumulation of memory B cells. In addition, we observed that Alum/TLR7 induces a recruitment of naïve antigen-specific B cells within the draining LN that may help to sustain the germinal center reaction. Our data further support Alum/TLR7 as a new promising adjuvant, which might contribute to meet the expectations of the GVAP for 2020 and beyond.

Vaccine adjuvant MF59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells.

MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs), that are facilitated to engulf antigen and transport it to draining lymph node (dLN) where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs). Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE) as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of vaccine adjuvants based on emulsions.

Human cytomegalovirus pUL10 interacts with leukocytes and impairs TCR-mediated T-cell activation.

Human cytomegalovirus (HCMV) is known to exert suppressive effects on the host immune system through expression of various viral genes, thus directly and indirectly affecting antiviral immunity of the infected individuals. We report here that HCMV UL10 encodes a protein (pUL10) with immunosuppressive properties. UL10 has been classified as a member of the HCMV RL11 gene family. Although pUL10 is known to be dispensable for viral replication in cultured cells, its amino-acid sequence is well conserved among different HCMV isolates, suggesting that the protein has a crucial role in viral survival in the host environment. We show that pUL10 is cleaved from the cell surface of fibroblasts as well as epithelial cells and interacts with a cellular receptor ubiquitously expressed on the surface of human leukocytes, demonstrated by ex vivo cell-based assays and flow cytometric analyses on both lymphoid cell lines and primary blood cells. Furthermore, preincubation of peripheral blood mononuclear cells with purified pUL10 ectodomain results in significantly impaired proliferation and substantially reduced pro-inflammatory cytokine production, in particular in CD4+ T cells upon in vitro T-cell stimulation. The inhibitory effect of pUL10 is also observed on antigen receptor-mediated intracellular tyrosine phosphorylation in a T-cell line. Based on these observations, we suggest that pUL10 is a newly identified immunomodulatory protein encoded by HCMV. Further elucidation of interactions between pUL10 and the host immune system during HCMV may contribute to finding ways towards new therapies for HCMV infection.

Ultrastructural Visualization of Vaccine Adjuvant Uptake In Vitro and In Vivo.

Adjuvants are substances that enhance adaptive immune responses when formulated in a vaccine. Alum and MF59 are two vaccine adjuvants licensed for human vaccination. Their mode of action has not been completely elucidated. Here we show the first ultrastructural visualization of Alum and MF59 interaction with immune cells in vitro and in vivo. We observed that Alum is engulfed by cells as inclusions of laminae that are detectable within draining lymph nodes. MF59 is instead engulfed by cells in vitro as low-electron-dense lipid-like inclusions that display a vesicle pattern, as confirmed by confocal microscopy using fluorescently labeled MF59. However, lipid-like inclusions with different high- and low-electron-dense content are detected within cells of draining lymph nodes when injecting MF59. As high-electron-dense lipid-like inclusions are also detected upon injection of Alum, our results suggest that the low-electron-dense inclusions are formed by engulfed MF59, whereas the high-electron-dense inclusions are proper lipid inclusions. Thus, we demonstrated that vaccine adjuvants are engulfed as inclusions by lymph node cells and hypothesize that adjuvant treatment may modify lipid metabolism.

Vaccine adjuvant MF59 promotes retention of unprocessed antigen in lymph node macrophage compartments and follicular dendritic cells.

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.

Surface molecules on stimulated plasmacytoid dendritic cells are sufficient to cross-activate resting myeloid dendritic cells.

Human plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells (mDCs) are 2 types of antigen-presenting cells that exert complementary roles in innate immune responses. We demonstrated previously that in the presence of suboptimal stimulation or when only 1 dendritic cell type is directly stimulated, contact-dependent crosstalk between mDCs and pDCs leads to the activation of both cell types and thus provides them with the ability to induce an optimal T-cell response. The precise mechanism is currently unknown. Here we demonstrate that pDCs, unable to secrete soluble factors because of previous stimulation, induce optimal mDC maturation, indicating that resting immature mDCs are fully competent to respond to Toll-like receptor-9-engaged pDCs in the absence of soluble factors. Thus, we conclude that immature mDCs already express receptors recognized by ligands that are upregulated on the surface of activated pDCs. Intercellular adhesion molecule-1 upregulated by activated pDCs may play a role in a donor-dependent manner.

Hepatitis C virions subvert natural killer cell activation to generate a cytokine environment permissive for infection.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is remarkably successful in establishing persistent infections due to its ability to evade host immune responses through a combination of mechanisms including modulation of interferon (IFN) signalling in infected cells, interference with effector cell function of the immune system and continual viral genetic variation. We have previously demonstrated that natural killer (NK) cells can be inhibited in vitro by recombinant HCV glycoprotein E2 via cross-linking of CD81, a cellular co-receptor for the virus. METHODS: Taking advantage of the recently established tissue-culture system for HCV, we have studied the effects of CD81 engagement by the HCV envelope glycoprotein E2 when the protein is part of complete, infectious viral particles. Specifically, we asked whether exposure to HCV viral particles (HCVcc) affects activation of NK cells and whether altered NK cell activation, in turn, impacts on HCV infectivity. RESULTS: We found that immobilized HCVcc, unlike soluble HCVcc, inhibited IFN-gamma production by interleukin (IL)-12 activated NK cells, and that this effect was mediated by engagement of cellular CD81 by HCV-virion displayed E2. In contrast, NK-production of IL-8 was increased in the presence of HCV. The cytokines produced by IL-12 activated NK cells strongly reduced the establishment of productive HCV infection. Importantly, NK-cell derived cytokines secreted in the presence of HCVcc showed a diminished antiviral effect that correlated with IFN-gamma reduction, while IL-8 concentrations had no impact on HCV infectivity. CONCLUSIONS: Exposure to HCVcc modulates the pattern of cytokines produced by NK cells, leading to reduced antiviral activity.

Human plasmacytoid dendritic cells are unresponsive to bacterial stimulation and require a novel type of cooperation with myeloid dendritic cells for maturation.

Dendritic cell (DC) populations play unique and essential roles in the detection of pathogens, but information on how different DC types work together is limited. In this study, 2 major DC populations of human blood, myeloid (mDCs) and plasmacytoid (pDCs), were cultured alone or together in the presence of pathogens or their products. We show that pDCs do not respond to whole bacteria when cultured alone, but mature in the presence of mDCs. Using purified stimuli, we dissect this cross-talk and demonstrate that mDCs and pDCs activate each other in response to specific induction of only one of the cell types. When stimuli for one or both populations are limited, they synergize to reach optimal activation. The cross-talk is limited to enhanced antigen presentation by the nonresponsive population with no detectable changes in the quantity and range of cytokines produced. We propose that each population can be a follower or leader in immune responses against pathogen infections, depending on their ability to respond to infectious agents. In addition, our results indicate that pDCs play a secondary role to induce immunity against human bacterial infections, which has implications for more efficient targeting of DC populations with improved vaccines and therapeutics.

Functional specialization of human circulating CD16 and CD1c myeloid dendritic-cell subsets.

Human blood contains 2 populations of dendritic cells (DCs): plasmacytoid and myeloid (mDC). mDCs are subdivided into 3 subsets using the surface markers CD16, CD1c, and BDCA-3. Their role as pathogen sentinels and adjuvant targets was tested by phenotypic and functional analysis. We show that mDC subsets are immature and express mRNA for most toll-like receptors (TLRs), except for TLR3 in CD16-mDCs. The most represented subsets, CD16- and CD1c-mDCs, are similarly responsive to all TLR agonists. Among 31 cytokines tested, both subsets produce CXCL8 (IL-8)/tumor necrosis factor-alpha (TNF-alpha)/IL-6/CCL3 (MIP-1 alpha)/CCL4 (MIP-1beta)/IL-1 beta. CXCL8 (IL-8) is the predominant cytokine produced by CD1c-mDCs on TLR engagement, whereas all other cytokines, particularly TNF-alpha, are secreted in 10-fold to 100-fold higher amounts by CD16-mDCs. CD16-mDCs cocultured with human umbilical vein endothelial cells induce a significantly higher production of CXCL10 (IP-10), granulocyte-macrophage colony-stimulating factor, and granulocyte colony-stimulating factor than CD1c-mDCs. In addition, interleukin-3 and type I interferons are stimuli specifically for DC maturation rather than cytokine secretion, whereas TNF-alpha is almost ineffective in inducing either function, suggesting a mechanism of T-cell-DC crosstalk and of rapid induction of antigen-presenting cell function during viral infection rather than inflammation. In conclusion, CD16-mDCs show strong proinflammatory activity, whereas CD1c-mDCs appear to be mainly inducers of chemotaxis.