Monocyte-derived dendritic cells in innate and adaptive immunity

Monocytes have been classically considered essential elements in relation with innate immune responses against pathogens, and inflammatory processes caused by external aggressions, infection and autoimmune disease. However, although their potential to differentiate into dendritic cells (DCs) was discovered 14 years ago, their functional relevance with regard to adaptive immune responses has only been uncovered very recently. Studies performed over the last years have revealed that monocyte-derived DCs play an important role in innate and adaptive immunity, due to their microbicidal potential, capacity to stimulate CD4(+) and CD8(+) T-cell responses and ability to regulate Immunoglobulin production by B cells. In addition, monocyte-derived DCs not only constitute a subset of DCs formed at inflammatory foci, as previously thought, but also comprise different subsets of DCs located in antigen capture areas, such as the skin and the intestinal, respiratory and reproductive tracts.     

Type I IFN-dependent T cell activation is mediated by IFN-dependent dendritic cell OX40 ligand expression and is independent of T cell IFNR expression

Type I IFNs are important for direct control of viral infection and generation of adaptive immune responses. Recently, direct stimulation of CD4(+) T cells via type I IFNR has been shown to be necessary for the formation of functional CD4(+) T cell responses. In contrast, we find that CD4(+) T cells do not require intrinsic type I IFN signals in response to combined TLR/anti-CD40 vaccination. Rather, the CD4 response is dependent on the expression of type I IFNR (IFNαR) on innate cells. Further, we find that dendritic cell (DC) expression of the TNF superfamily member OX40 ligand was dependent on type I IFN signaling in the DC, resulting in a reduced CD4(+) T cell response that could be substantially rescued by an agonistic Ab to the receptor OX40. Taken together, we show that the IFNαR dependence of the CD4(+) T cell response is accounted for exclusively by defects in DC activation.     

ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway

Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.     

Pathogen-induced private conversations between natural killer and dendritic cells

Natural killer (NK) cells and dendritic cells (DCs) are recruited to inflammatory tissues in response to infection. Following priming by pathogen-derived products, their reciprocal interactions result in a potent activating crosstalk that regulates both the quality and the intensity of innate immune responses. Thus, pathogen-primed NK cells, in the presence of cytokines released by DCs, become activated. At this stage they favor DC maturation and also select the most suitable DCs for subsequent migration to lymph nodes and priming of T cells. In addition, a specialized subset of NK cells might directly participate in the process of T-cell priming via the release of interferon (IFN)gamma. Thus, the reciprocal crosstalk between NK cells and DCs that is induced by microbial products not only promotes rapid innate responses against pathogens but also favor the generation of appropriate downstream adaptive responses.     

IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1^−/− mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1^−/− mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection.     

Sphingosine analogue AAL-R increases TLR7-mediated dendritic cell responses via p38 and type I IFN signaling pathways

Sphingosine analogues display immunosuppressive activities and thus have therapeutic potential in the treatment of autoimmune diseases. In this study, we investigated the effects of the sphingosine analogue AAL-R (FTY720 derivative) on dendritic cell (DC) response upon TLR stimulation. Unlike its known immunosuppressive activity, AAL-R increased TLR7-mediated DC responses by elevating the levels of MHC class I and costimulatory molecules and type I IFN expression and by enhancing the capacity of DCs to induce CD8(+) T cell proliferation. Importantly, the stimulatory activity of AAL-R was dependent on type I IFN signaling, as type I IFN receptor-deficient DCs failed to respond to AAL-R. Also, AAL-R activated p38 MAPK to increase type I IFN synthesis and TLR7-mediated DC maturation. These findings enhance our understanding of sphingosine regulation of the host immune system, in particular upon pathogenic infections.     

Variable requirement of dendritic cells for recruitment of NK and T cells to different TLR agonists

TLRs initiate the host immune response to microbial pathogens by activating cells of the innate immune system. Dendritic cells (DCs) can be categorized into two major groups, conventional DCs (including CD8(+) and CD8(-) DCs) and plasmacytoid DCs. In mice, these subsets of DCs express a variety of TLRs, with conventional DCs responding in vitro to predominantly TLR3, TLR4, TLR5, and TLR9 ligands, and plasmacytoid DCs responding mainly to TLR7 and TLR9 ligands. However, the in vivo requirement of DCs to initiate immune responses to specific TLR agonists is not fully known. Using mice depleted of >90% of CD11c(+) MHC class II(+) DCs, we demonstrate that cellular recruitment, including CD4(+) T cell and CX5(+)DX5(+) NK cell recruitment to draining lymph nodes following the footpad administration of TLR4 and TLR5 agonists, is dramatically decreased upon reduction of DC numbers, but type I IFN production can partially substitute for DCs in response to TLR3 and TLR7 agonists. Interestingly, TLR ligands can activate T cells and NK cells in the draining lymph nodes, even with reduced DC numbers. The findings reveal considerable plasticity in the response to TLR agonists, with TLR4 and TLR5 agonists sharing the requirement of DCs for subsequent lymph node recruitment of NK and T cells.     

Usp18 promotes conventional CD11b+ dendritic cell development

Dendritic cells (DCs) represent the key cells linking innate and adaptive immune responses. It is critical to understand the molecular factors regulating DC differentiation. Usp18 is an IFN-inducible member of the ubiquitin-specific protease family, which deconjugates ubiquitin-like modifier ISG15 from target proteins and competitively inhibits IFN-α/β-induced JAK/STAT activation. This study demonstrates that the frequency of conventional CD11b(+) DCs in the spleen of Usp18(-/-) mice was significantly reduced, whereas the frequencies of conventional CD8(+) DCs and plasmacytoid DCs remained normal. In addition, Usp18(-/-) bone marrow (BM) cells generate DCs less efficiently in GM-CSF-supplemented culture, demonstrating a fundamental defect throughout the DC differentiation pathway. Usp18(-/-) BM cells were rescued by exogenous expression of either wild-type or deconjugation-inactive Usp18, and superimposition of an IFN-α/β receptor knockout returned in vivo DC populations to normal, clearly showing that the defect seen is due solely to Usp18's effect on IFN signaling. Finally, Usp18(-/-) BM-derived DCs expressed high levels of SOCS1/SOCS3, known inhibitors of GM-CSF signaling, providing a mechanistic explanation for the phenotype. In conclusion, we have identified a novel role of Usp18 in modulating conventional CD11b(+) DC development via its inhibitory effect on type I IFN signaling.     

The Timing of IFNβ Production Affects Early Innate Responses to Listeria monocytogenes and Determines the Overall Outcome of Lethal Infection

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a crucial role in the first phase of host defense against infections and tumors. Listeria monocytogenes (Lm) is an intracellular pathogen that colonizes the cytosol of eukaryotic cells. Recent findings have shown Lm specifically in splenic CD8a(+) DCs shortly after intravenous infection. We examined gene expression profiles of mouse DCs exposed to Lm to elucidate the molecular mechanisms underlying DCs interaction with Lm. Using a functional genomics approach, we found that Lm infection induced a cluster of late response genes including type I IFNs and interferon responsive genes (IRGs) in DCs. Type I INFs were produced at the maximal level only at 24 h post infection indicating that the regulation of IFNs in the context of Lm infection is delayed compared to the rapid response observed with viral pathogens. We showed that during Lm infection, IFNγ production and cytotoxic activity were severely impaired in NK cells compared to E. coli infection. These defects were restored by providing an exogenous source of IFNβ during the initial phase of bacterial challenge. Moreover, when treated with IFNβ during early infection, NK cells were able to reduce bacterial titer in the spleen and significantly improve survival of infected mice. These findings show that the timing of IFNβ production is fundamental to the efficient control of the bacterium during the early innate phase of Lm infection.     

Dendritic cells in innate immune responses against HIV

Dendritic cells (DCs) were recently found to be innate immunity effectors against tumoral cells and viruses. (i) In response to most viruses, including HIV, plasmacytoid DCs are responsible for most of the type I IFN secretion, which is strongly anti-viral and induces TH1 type responses. Myeloid DCs secrete IL-12, which is also important for TH1-type and cytotoxic responses. In HIV patient blood, both DC population numbers decrease as early as the primary stage. Plasmacytoid DC numbers correlate with type I IFN secretion, which is a prognosis predictor, particularly under treatment. IL-12 secretion is also defective. Immunotherapies to replace the defective cytokines or to restore a potentially defective DC-T lymphocyte feed-back might help patients restore their immune responses under antiviral therapy. (ii) After measles and other viral infections, or incubation with dsRNA, DCs become cytotoxic and consequently exhibit natural killer function, through upregulation of type I IFN secretion which enhances TRAIL expression. In HIV infection, this mechanism was not demonstrated yet, but it might a) be responsible for the massive apoptosis of uninfected lymphocytes, and b) increase specific immunity through cross-presentation of antigens from infected cells killed by DCs. (iii) DCs direct expansion and effector functions of NK cells in the absence of adaptive-type cytokines and modulate NKT cell IFN-gamma production. Reciprocally, NK activation triggers DC maturation. HIV-1 Tat inhibits NK cell cytotoxicity directly and probably through inhibition of IL-12 secretion by DC. Therefore, understanding the functions of DCs in innate immune responses and in pathogenesis will help obtain better HIV replication control.     

HSP110-HER2/neu chaperone complex vaccine induces protective immunity against spontaneous mammary tumors in HER-2/neu transgenic mice

Heat shock proteins (HSPs) are shown to be strong immunoadjuvants, eliciting both innate and adaptive immune responses against cancers. HSP110 is related in sequence to HSP70 and is approximately 4-fold more efficient in binding to and stabilizing denatured protein substrates compared with HSP70. In the present study we evaluated the ability of a heat shock complex of HSP110 with the intracellular domain (ICD) of human HER-2/neu to elicit effective antitumor immune responses and to inhibit spontaneous mammary tumors in FVB-neu (FVBN202) transgenic mice. The HSP110-ICD complex was capable of breaking tolerance against the rat neu protein and inhibiting spontaneous mammary tumor development. This vaccine induced ICD-specific IFN-gamma and IL-4 production. Depletion studies revealed that CD8(+) T cells were involved in protection against challenge with mouse mammary tumors, whereas CD4(+) T cells revealed partial protection. Increased IgG2a Ab titer in the sera of tumor-free animals after vaccination and elevated CD4(+) CD25(+) regulatory T cells in the PBL of tumor-bearing animals suggested that IFN-gamma-producing Th1 cells may be responsible for partial protection of CD4(+) T cells against the mammary tumor challenge, whereas CD4(+)CD25(+) regulatory T cells (Th2 cells) may suppress the antitumor immune responses. Together, these results suggest that HSP110-ICD complex can elicit effective IFN-gamma-producing T cells against spontaneous mammary tumors and that up-regulation of CD4(+) CD25(+) regulatory T cells may prevent complete eradication of the tumor following immunotherapy.     

Herpes simplex viruses and induction of interferon responses

Herpes simplex viruses (HSV) are human pathogens responsible for a variety of diseases,including localized mucocutaneous lesions,encephalitis,and disseminated diseases.HSV infection leads to rapid induction of innate immune responses.A critical part of this host response is the type I IFN system including the induction of type I IFNs,IFN-mediated signaling and amplification of IFN response.This provides the host with immediate countermeasure during acute infection to limit initial viral replication and to facilitate an appropriate adaptive immune response.However,HSV has devised multiple strategies to evade and interfere with innate immunity.This review will focus on the induction of type I IFN response by HSV during acute infection and current knowledge of mechanisms by which HSV interferes with this induction process.     

Immunomodulatory functions of type I interferons

Interferon-α (IFNα) and IFNβ, collectively known as type I IFNs, are the major effector cytokines of the host immune response against viral infections. However, the production of type I IFNs is also induced in response to bacterial ligands of innate immune receptors and/or bacterial infections, indicating a broader physiological role for these cytokines in host defence and homeostasis than was originally assumed. The main focus of this Review is the underappreciated immunomodulatory functions of type I IFNs in health and disease. We discuss their function in the regulation of innate and adaptive immune responses, the response to bacterial ligands, inflammasome activation, intestinal homeostasis and inflammatory and autoimmune diseases.     

CD8+ T-cell priming regulated by cytokines of the innate immune system

Host protection against a variety of pathogens and tumours requires the efficient induction of CD8(+) T-cell responses. Yet, it has proven difficult to develop vaccines that effectively stimulate this type of cellular immunity. One well-defined obstacle is antigen accessibility to the MHC class I processing pathway. However, cytokines that are produced by cells of the innate immune system also have a key role in CD8(+) T-cell responses, by enhancing 'cross-presentation' and/or inducing CD8(+) T-cell priming and differentiation. Here, we discuss how innate cytokine responses regulate CD8(+) T-cell immunity, and argue that a greater understanding of these processes will be essential for effective tailoring of vaccine-induced cellular immune responses.     

Human metapneumovirus glycoprotein G inhibits TLR4-dependent signaling in monocyte-derived dendritic cells

Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory infections in children and adults. Recent work from our group demonstrated that hMPV G glycoprotein is an important virulence factor, responsible for inhibiting innate immune responses in airway epithelial cells. Myeloid dendritic cells (DCs) are potent APCs and play a major role in initiating and modulating the innate and adaptive immune responses. In this study, we found that TLR4 plays a major role in hMPV-induced activation of monocyte-derived DCs (moDCs), as downregulation of its expression by small interfering RNA significantly blocked hMPV-induced chemokine and type I IFN expression. Similar results were found in bone marrow-derived DCs from TLR4-deficient mice. moDCs infected with a virus lacking G protein expression produced higher levels of cytokines and chemokines compared with cells infected with wild-type virus, suggesting that G protein plays an inhibitory role in viral-induced cellular responses. Specifically, G protein affects TLR4-dependent signaling, as infection of moDCs with recombinant hMPV lacking G protein inhibited LPS-induced production of cytokine and chemokines significantly less than did wild-type virus, and treatment of moDCs with purified G protein resulted in a similar inhibition of LPS-dependent signaling. Our results demonstrate that hMPV G protein plays an important role in inhibiting host innate immune responses, likely affecting adaptive responses too.     

Elimination of tumor by CD47/PD-L1 dual-targeting fusion protein that engages innate and adaptive immune responses

The host immune system generally serves as a barrier against tumor formation. Programmed death-ligand 1 (PD-L1) is a critical “don't find me” signal to the adaptive immune system, whereas CD47 transmits an anti-phagocytic signal, known as the “don't eat me” signal, to the innate immune system. These and similar immune checkpoints are often overexpressed on human tumors. Thus, dual targeting both innate and adaptive immune checkpoints would likely maximize anti-tumor therapeutic effect and elicit more durable responses. Herein, based on the variable region of atezolizumab and consensus variant 1 (CV1) monomer, we constructed a dual-targeting fusion protein targeting both CD47 and PD-L1 using “Knobs-into-holes” technology, denoted as IAB. It was effective in inducing phagocytosis of tumor cells, stimulating T-cell activation and mediating antibody-dependent cell-mediated cytotoxicity in vitro. No obvious sign of hematological toxicity was observed in mice administered IAB at a dose of 100 mg/kg, and IAB exhibited potent antitumor activity in an immune-competent mouse model of MC38. Additionally, the anti-tumor effect of IAB was impaired by anti-CD8 antibody or clodronate liposomes, which implied that both CD8+ T cells and macrophages were required for the anti-tumor efficacy of IAB and IAB plays an essential role in the engagement of innate and adaptive immune responses. Collectively, these results demonstrate the capacity of an elicited endogenous immune response against tumors and elucidate essential characteristics of synergistic innate and adaptive immune response, and indicate dual blockade of CD47 and PD-L1 by IAB may be a synergistic therapy that activates both innate and adaptive immune response against tumors.     

A temporal role of type I interferon signaling in CD8+ T cell maturation during acute West Nile virus infection

A genetic absence of the common IFN-α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR(-/-) mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8(+) T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8(+) T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8(+) T cell development requires type I IFN signaling. WNV infection experiments in BATF3(-/-) mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8(+) T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8(+) T cell response at a stage distinct from the initial priming event.     

CD11c controls herpes simplex virus 1 responses to limit virus replication during primary infection

CD11c is expressed on the surface of dendritic cells (DCs) and is one of the main markers for identification of DCs. DCs are the effectors of central innate immune responses, but they also affect acquired immune responses to infection. However, how DCs influence the efficacy of adaptive immunity is poorly understood. Here, we show that CD11c(+) DCs negatively orchestrate both adaptive and innate immunity against herpes simplex virus type 1 (HSV-1) ocular infection. The effectiveness and quantity of virus-specific CD8(+) T cell responses are increased in CD11c-deficient animals. In addition, the levels of CD83, CD11b, alpha interferon (IFN-α), and IFN-β, but not IFN-γ, were significantly increased in CD11c-deficient animals. Higher levels of IFN-α, IFN-β, and CD8(+) T cells in the CD11c-deficient mice may have contributed to lower virus replication in the eye and trigeminal ganglia (TG) during the early period of infection than in wild-type mice. However, the absence of CD11c did not influence survival, severity of eye disease, or latency. Our studies provide for the first time evidence that CD11c expression may abrogate the ability to reduce primary virus replication in the eye and TG via higher activities of type 1 interferon and CD8(+) T cell responses.