Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

Regulation of dendritic cell function by microbial stimuli.

Dendritic cells (DC) initiate T cell responses and produce cytokines and other molecules that can regulate the class adaptive immunity. It is increasingly clear that DC in vivo are in a "resting" state and require exogenous signals to transit into an "effector" state in which they can prime T cells. Much of this DC activation process appears to be regulated by infection. Exposure of murine DC to certain pathogens or their products triggers DC migration to T cell areas of secondary lymphoid tissues, improves MHC presentation and increases DC co-stimulatory potential. Pathogen recognition can also initiate cytokine production and/or condition DC to produce cytokines in response to subsequent T cell feedback signals delivered via CD40 and similar receptors. Recognition of pathogens by DC is largely dependent on Toll-like receptors (TLRs). Interestingly, mouse splenic CD8alpha+ and CDalpha-CD4- DC have the ability to produce either IL-12 p70 or IL-10 depending on the nature of the pathogen encountered. In contrast, CD4+ DC seem incapable of producing IL-12 p70. Thus, the nature of the pathogen can dictate the type of cytokine that is made by some DC subsets, allowing them to prime distinct types of immune responses. Overall, DC display significant plasticity in their ability to respond to infection and direct adaptive immunity.

Lipoxin-mediated inhibition of IL-12 production by DCs: a mechanism for regulation of microbial immunity.

Lipoxins are eicosanoid mediators that show potent inhibitory effects on the acute inflammatory process. We show here that the induction of lipoxin A(4) (LXA(4)) accompanied the in vivo suppression of interleukin 12 (IL-12) responsiveness of murine splenic dendritic cells (DCs) after microbial stimulation with an extract of Toxoplasma gondii. This paralysis of DC function could not be triggered in mice that were deficient in a key lipoxygenase involved in LXA(4) biosynthesis. In addition, DCs pre-treated with LXA(4) became refractory to microbial stimulation for IL-12 production in vitro and mice injected with a stable LXA(4) analog showed reduced splenic DC mobilization and IL-12 responses in vivo. Together, these findings indicate that the induction of lipoxins in response to microbial stimulation can provide a potent mechanism for regulating DC function during the innate response to pathogens.

IL-12 induction by a TH1-inducing adjuvant in vivo: dendritic cell subsets and regulation by IL-10.

IL-12 induction is critical for immune responses against many viruses and intracellular bacterial pathogens. Recent studies suggest that IL-12-secreting dendritic cells (DC) are potent Th1-inducing APC. However, controversy exists concerning the function of DC subsets. Murine studies have suggested that CD8(+) DC preferentially induce Th1 responses, whereas CD8(-) DC induce Th2 development; in this model, different DC subsets prime different responses. Alternatively, the propensity of DC subsets to prime a Th1 response could depend upon the type of initial stimulus. We used a prototypic Th1-inducing adjuvant, heat-killed Brucella abortus (HKBA) to assess stimulation of DC subsets, relationship between Ag burden and IL-12 production, and down-regulation of DC subset IL-12 production by IL-10. In this study, we show that DC were sole producers of IL-12, although most HKBA uptake was by splenic macrophages and granulocytes. More CD8(-) than CD8(+) DC produced IL-12 after HKBA challenge, whereas only CD8(+) DC produced IL-12 after injection of another Th1-promoting microbial substance, soluble Toxoplasma gondii Ags. Studies in IL-10-deficient mice revealed that IL-10 down-regulates frequency and duration of IL-12 production by both DC subsets. In the absence of IL-10, IL-12 expression is enabled in CD11c(low) cells, but not in macrophages or granulocytes. These findings support the concept of DC as the major IL-12 producers in spleens, but challenge the notion that CD8(+) and CD8(-) DC are destined to selectively induce Th1 or Th2 responses, respectively. Thus, the nature of the stimulating substance is important in determining which DC subsets are activated to produce IL-12.

CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal.

CD40 ligation triggers IL-12 production by dendritic cells (DC) in vitro. Here, we demonstrate that CD40 cross-linking alone is not sufficient to induce IL-12 production by DC in vivo. Indeed, resting DC make neither the IL-12 p35 nor IL-12 p40 subunits and express only low levels of CD40. Nevertheless, after DC activation by microbial stimuli that primarily upregulate IL-12 p40 and augment CD40 expression, CD40 ligation induces a significant increase in IL-12 p35 and IL-12 p70 heterodimer production. Similarly, IL-12 p70 is produced during T cell activation in the presence but not in the absence of microbial stimuli. Thus, production of bioactive IL-12 by DC can be amplified by T cell-derived signals but must be initiated by innate signals.

Microbial and T cell-derived stimuli regulate antigen presentation by dendritic cells in vivo.

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4(+) T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8alpha(+) DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.

The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli.

During their final differentiation or maturation, dendritic cells (DCs) redistribute their major histocompatibility complex (MHC) class II products from intracellular compartments to the plasma membrane. Using cells arrested in the immature state, we now find that DCs also regulate the initial intracellular formation of immunogenic MHC class II-peptide complexes. Immature DCs internalize the protein antigen, hen egg lysozyme (HEL), into late endosomes and lysosomes rich in MHC class II molecules. There, despite extensive colocalization of HEL protein and MHC class II products, MHC class II-peptide complexes do not form unless the DCs are exposed to inflammatory mediators such as tumor necrosis factor alpha, CD40 ligand, or lipoplolysaccharide. The control of T cell receptor (TCR) ligand formation was observed using the C4H3 monoclonal antibody to detect MHC class II-HEL peptide complexes by flow cytometry and confocal microscopy, and with HEL-specific 3A9 transgenic T cells to detect downregulation of the TCR upon MHC-peptide encounter. Even the binding of preprocessed HEL peptide to MHC class II is blocked in immature DCs, including the formation of C4H3 epitope in MHC class II compartments, suggesting an arrest to antigen presentation at the peptide-loading step, rather than an enhanced degradation of MHC class II-peptide complexes at the cell surface, as described in previous work. Therefore, the capacity of late endosomes and lysosomes to produce MHC class II-peptide complexes can be strictly controlled during DC differentiation, helping to coordinate antigen acquisition and inflammatory stimuli with formation of TCR ligands. The increased ability of maturing DCs to load MHC class II molecules with antigenic cargo contributes to the >100-fold enhancement of the subsequent primary immune response observed when immature and mature DCs are compared as immune adjuvants in culture and in mice.

CCR5 provides a signal for microbial induced production of IL-12 by CD8 alpha+ dendritic cells.

The activation of dendritic cells (DC) to produce interleukin 12 (IL-12) is thought to be a key step in the initiation of cell-mediated immunity to intracellular pathogens. Here we show that ligation of the C-C chemokine receptor (CCR) 5 can provide a major signal for the induction of IL-12 synthesis by the CD8 alpha+ subset of DC and that this pathway is important in establishing interferon gamma-dependent resistance to the protozoan parasite Toxoplasma gondii. These findings support the concept that the early induction of chemokines by invading pathogens is a critical step not only for the recruitment of DC but also for the determination of their subsequent immunologic function.

Paralysis of dendritic cell IL-12 production by microbial products prevents infection-induced immunopathology.

Interleukin-12 plays a major role in immunity to intracellular pathogens by governing the development of IFNgamma-dependent host resistance. Nevertheless, unregulated IL-12 synthesis can lead to immunopathology, an outcome prevented by the concurrent expression of interleukin-10. Dendritic cells (DC) are an important source of the initial IL-12 stimulated by microbial agents. Here, we show that, following systemic triggering, DC can no longer be restimulated to produce IL-12 in vivo while continuing to respond in vitro. When infected with Toxoplasma gondii during this refractory state, mice mount impaired acute IFNgamma responses and, in the case of IL-10-deficient animals, are protected from cytokine-induced mortality. These findings demonstrate a previously unrecognized form of immunologic paralysis involving DC that can protect from infection-induced immunopathology.

The role of dendritic cells in the induction and regulation of immunity to microbial infection.

Dendritic cells (DCs) play a critical role in the initiation and regulation of immune responses. Recent advances have begun to uncover the nature and diversity of DC-pathogen interactions and the modulation of DC function by microbial stimuli. Antigen pulsed DCs have also been shown in several infection models to induce high levels of protective immunity and to display immunotherapeutic potential. The study of the function of DCs in the response to infection is thus an exciting and rapidly expanding field with important implications for both fundamental and clinical immunology.

Analysis of adjuvant function by direct visualization of antigen presentation in vivo: endotoxin promotes accumulation of antigen-bearing dendritic cells in the T cell areas of lymphoid tissue.

T cell activation requires exposure to processed Ag and signaling by cytokines and costimulatory ligands. Adjuvants are thought to enhance immunity primarily through up-regulation of the latter signals. Here, we explore the effect of the bacterial adjuvant, endotoxin, on Ag presentation by B cells and dendritic cells (DC). Using an mAb (C4H3) specific for the hen egg lysozyme (HEL) 46-61 determinant bound to I-Ak, we analyze processed Ag expression and the tissue distribution of presenting cells following systemic administration of soluble HEL to mice. In both LPS-responsive and -hyporesponsive mice given endotoxin-containing HEL, B cells rapidly display surface 46-61/I-Ak complexes. In marked contrast, in LPS-hyporesponsive mice, splenic DC show little gain in C4H3 staining. In LPS-responsive animals, interdigitating DC in T cell areas show no staining above background at early times after HEL administration, but C4H3+ DC rapidly accumulate in the outer periarteriolar lymphoid sheaths (PALS) and in follicular areas. Within a few hours, C4H3+ DC appear in the T cell areas, concomitant with a decline in C4H3+ cells in the outer PALS, suggesting migration between these two sites. Endotoxin enhancement of C4H3 staining is seen for both CD8alpha- and CD8alpha+ DC subsets. These data suggest that a major effect of adjuvants is to promote mobilization of Ag-bearing DC to the T areas of lymphoid tissue, and possibly also to enhance Ag processing by these DC. Thus, microbial products promote T cell immunity not only through DC activation for cosignaling, but through improvement in signal 1 delivery.

Ignition of the type 1 response to intracellular infection by dendritic cell-derived interleukin-12.

Host resistance to many intracellular pathogens is dependent on the induction of host IFN-gamma. This response in turn is triggered by the critical initiation cytokine, IL-12. Activated macrophages provide a major source of IL-12 during infection yet are unlikely to be the initial cell to produce the cytokine because of their need for IFN-gamma priming and/or other co-stimulatory signals. We have utilized an in vivo approach to identify the primary IL-12 producing cells which respond to the intracellular protozoan Toxoplasma gondii. Our results indicate that in spleen interdigitating dendritic cells (IDC) but not macrophages rapidly synthesize IL-12 after injection of parasite products or live tachyzoites. This response is both IFN-gamma and T lymphocyte independent. The same microbial stimulus results in the migration of IDC precursors into T cell areas and the upregulation of co-stimulatory cell-surface molecules. We postulate that these early dendritic cell activation events represent the "ignition switch" for the subsequent Type 1 cytokine response which leads to control of infection.

Differential TCR signaling regulates apoptosis and immunopathology during antigen responses in vivo.

Clonal selection theories postulate that lymphocyte fate is regulated by antigen receptor specificity. However, lymphocyte apoptosis is induced through nonantigen-specific receptors such as Fas (CD95/APO-1) or TNFR. We define a selective TCR that controls apoptosis by Fas or TNFR stimulation. Variant ligands can deliver this "competence to die" signal without the full TCR signals necessary for cytokine synthesis. These partial agonists regulate T cell deletion in vivo even when Fas or TNF is provided by T cells of unrelated specificity, but they do not cause the liver necrosis that is associated with T cell elimination by the full agonist. Thus, selective signaling ligands regulate T cell deletion and immune damage in vivo and may be important for peripheral T cell tolerance.

Selective induction of apoptosis in mature T lymphocytes by variant T cell receptor ligands.

Activation, anergy, and apoptosis are all possible outcomes of T cell receptor (TCR) engagement. The first leads to proliferation and effector function, whereas the others can lead to partial or complete immunological tolerance. Structural variants of immunizing peptide-major histocompatibility complex molecule ligands that induce selective lymphokine secretion or anergy in mature T cells in association with altered intracellular signaling events have been described. Here we describe altered ligands for mature mouse CD4(+) T helper 1 cells that lead to T cell apoptosis by the selective expression of Fas ligand (FasL) and tumor necrosis factor (TNF) without concomitant IL-2, IL-3, or interferon gamma production. All ligands that stimulated cell death were found to induce FasL and TNF mRNA expression and TCR aggregation ("capping") at the cell surface, but did not elicit a common pattern of tyrosine phosphorylation of the TCR-associated signal transduction chains. Thus, TCR ligands that uniquely trigger T cell apoptosis without inducing cytokines that are normally associated with activation can be identified.

Antigen-unspecific B cells and lymphoid dendritic cells both show extensive surface expression of processed antigen-major histocompatibility complex class II complexes after soluble protein exposure in vivo or in vitro.

Intravenous (i.v.) injection of high amounts of soluble proteins often results in the induction of antigen-specific tolerance or deviation to helper rather than inflammatory T cell immunity. It has been proposed that this outcome may be due to antigen presentation to T cells by a large cohort of poorly costimulatory or IL-12-deficient resting B cells lacking specific immunoglobulin receptors for the protein. However, previous studies using T cell activation in vitro to assess antigen display have failed to support this idea, showing evidence of specific peptide-major histocompatibility complex (MHC) class II ligand only on purified dendritic cells (DC) or antigen-specific B cells isolated from protein injected mice. Here we reexamine this question using a recently derived monoclonal antibody specific for the T cell receptor (TCR) ligand formed by the association of the 46-61 determinant of hen egg lysozyme (HEL) and the mouse MHC class II molecule I-Ak. In striking contrast to conclusions drawn from indirect T cell activation studies, this direct method of TCR ligand analysis shows that i.v. administration of HEL protein results in nearly all B cells in lymphoid tissues having substantial levels of HEL 46-61-Ak complexes on their surface. DC readily isolated from spleen also display this TCR ligand on their surface. Although the absolute number of displayed ligands is greater on such DC, the relative specific ligand expression compared to total MHC class II levels is similar or greater on B cells. These results demonstrate that in the absence of activating stimuli, both lymphoid DC and antigen-unspecific B cells present to a similar extent class II-associated peptides derived from soluble proteins in extracellular fluid. The numerical advantage of the TCR ligand-bearing B cells may permit them to interact first or more often with naive antigen-specific T cells, contributing to the induction of high-dose T cell tolerance or immune deviation.