Roman Spörri

Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4 + T cell populations lacking helper function

Dendritic cells (DCs) can be activated directly by triggering of receptors for pathogens or, indirectly, by exposure to inflammatory signals. It remains unclear, however, whether the two pathways result in qualitatively similar DCs or lead to equivalent adaptive immune responses. Here we report that indirect activation by inflammatory mediators generated DCs that supported CD4+ T cell clonal expansion but failed to direct T helper cell differentiation. In contrast, exposure to pathogen components resulted in fully activated DCs that promoted T helper responses. These results indicate that inflammation cannot substitute for contact with pathogen components in DC activation and suggest that the function of pattern recognition by DCs is to couple the quality of the adaptive immune response to the nature of the pathogen.

Inflammatory signals in dendritic cell activation and the induction of adaptive immunity

Summary:  Pathogen invasion induces a rapid inflammatory response initiated through the recognition of pathogen‐derived molecules by pattern recognition receptors (PRRs) expressed on both immune and non‐immune cells. The initial wave of pro‐inflammatory cytokines and chemokines limits pathogen spread and recruits and activates immune cells to eradicate the invaders. Dendritic cells (DCs) are responsible for initiating a subsequent phase of immunity, dominated by the action of pathogen‐specific T and B cells. As for the early pro‐inflammatory response, DC activation is triggered by PRR signals. These signals convert resting DCs into potent antigen‐presenting cells capable of promoting the expansion and effector differentiation of naive pathogen‐specific T cells. However, it has been argued that signals from PRRs are not a prerequisite for DC activation and that pro‐inflammatory cytokines have the same effect. Although this may appear like an efficient way to expand the number of DCs that initiate adaptive immunity, evidence is accumulating that DCs activated indirectly by inflammatory cytokines are unable to induce functional T‐cell responses. Here, we review the differences between PRR‐triggered and cytokine‐induced DC activation and speculate on a potential role for DCs activated by inflammatory signals in tolerance induction rather than immunity.

Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8α+ and CD8α−CD4− DC to make IL-12 p70. In contrast, exposure of CD8α+, CD4+ and CD8α−CD4− DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

MyD88-Dependent IFN-γ Production by NK Cells Is Key for Control of Legionella pneumophila Infection

Legionella pneumophila (Lpn) is a ubiquitous Gram-negative bacterium in aquatic systems and an opportunistic intracellular pathogen in immunocompromised humans causing a severe pneumonia known as Legionnaires’ disease. Using a mouse model, we investigated molecular and cellular players in the innate immune response to infection with Lpn. We observed robust levels of inflammatory cytokines in the serum upon intranasal or i.v. infection with live, virulent Lpn, but not with inactivated or avirulent bacteria lacking the Icm/Dot type IV secretion system. Interestingly, Lpn-induced serum cytokines were readily detectable regardless of the capacity of Icm/Dot-proficient Lpn to replicate in host cells and the Lpn permissiveness of the host mice. We found NK cell-derived IFN-γ to be the key cytokine in the resolution of Lpn infection, whereas type I IFNs did not appear to play a major role in our model. Accordingly, NK cell-depleted or IFN-II-R-deficient mice carried severely increased bacterial burdens or failed to control Lpn infection, respectively. Besides the dependence of inflammatory cytokine induction on Lpn virulence, we also demonstrate a strict requirement of MyD88 for this process, suggesting the involvement of TLRs in the recognition of Lpn. However, screening of several TLR-deficient hosts did not reveal a master TLR responsible for the sensing of an Lpn infection, but provided evidence for either redundancy of individual TLRs in Lpn recognition or TLR-independent induction of inflammatory responses.

A Novel Role for Neutrophils As Critical Activators of NK Cells

Neutrophils are essential players in innate immune responses to bacterial infection. Despite the striking resistance of Legionella pneumophila (Lpn) to bactericidal neutrophil function, neutrophil granulocytes are important effectors in the resolution of legionellosis. Indeed, mice depleted of neutrophils were unable to clear Lpn due to a lack of the critical cytokine IFN-γ, which is produced by NK cells. We demonstrate that this can be ascribed to a previously unappreciated role of neutrophils as major NK cell activators. In response to Lpn infection, neutrophils activate caspase-1 and produce mature IL-18, which is indispensable for the activation of NK cells. Furthermore, we show that the IL-12p70 response in Lpn-infected neutropenic mice is also severely reduced and that the Lpn-induced IFN-γ production by NK cells is strictly dependent on IL-12. However, since dendritic cells, and not neutrophils, are the source of Lpn-induced IL-12, its paucity is a consequence of the absence of IFN-γ produced by NK cells rather than the absence of neutrophils per se. Therefore, neutrophil-derived IL-18, in combination with dendritic cell-produced IL-12, triggers IFN-γ synthesis in NK cells in Lpn-infected mice. We propose a novel central role for neutrophils as essential IL-18 producers and hence NK cell “helpers” in bacterial infection.

Direct activation of antigen-presenting cells is required for CD8+ T-cell priming and tumor vaccination

Successful priming of adaptive immune responses is crucially dependent on innate activation signals that convert resting antigen-presenting cells (APCs) into immunogenic ones. APCs expressing the relevant innate pattern recognition receptors can be directly activated by pathogen-associated molecular patterns (PAMPs) to become competent to prime T-cell responses. Alternatively, it has been suggested that APCs could be activated indirectly by proinflammatory mediators synthesized by PAMP-exposed cells. However, data obtained with CD4+ T cells suggest that inflammatory signals often cannot substitute for direct pattern recognition in APC activation for the priming of T helper responses. To test whether the same is true for CD8+ T cells, we studied cytotoxic T lymphocyte development in vitro and in mixed chimeric mice in which coexisting APCs can either present a preprocessed model antigen or directly recognize a given PAMP, but not both. We show that indirectly activated APCs promote antigen-specific proliferation of naïve CD8+ T cells but fail to support their survival and cytotoxic T lymphocyte differentiation. Furthermore, CD8+ T cells primed by indirectly activated APCs are unable to reject tumors. Thus, inflammation cannot substitute for direct recognition of single PAMPs in CD8+ T-cell priming. These findings have important practical implications for vaccine design, indicating that adjuvants must be judiciously chosen to trigger the relevant pattern recognition receptors in APCs.

Newly Activated T Cells Promote Maturation of Bystander Dendritic Cells but Not IL-12 Production

The activation of dendritic cells (DC) leads to increased costimulatory activity (termed DC maturation) and, in some instances, production of immunomodulatory cytokines such as IL-12. Both innate and T cell-derived signals can promote DC activation but it is unclear to what extent the two classes of stimuli are interchangeable or regulate distinct aspects of DC function. In this study, we show that signals from newly activated CD4+ T cells cannot initiate IL-12 synthesis although they can amplify secretion of bioactive IL-12 p70 by DC exposed to an appropriate innate stimulus. This occurs exclusively in cis and does not influence IL-12 synthesis by bystander DC that do not present Ag. In marked contrast, signals from newly activated CD4+ T cells can induce an increase in DC costimulatory activity in the absence of any innate priming. This occurs both in cis and in trans, affecting all DC in the microenvironment, including those that do not bear specific Ag. Consistent with the latter, we show that newly activated CD4+ T cells in vivo can deliver “help” in trans, effectively lowering the number of MHC/peptide complexes required for proliferation of third-party naive CD4+ T cells recognizing Ag on bystander DC. These results demonstrate that DC maturation and cytokine production are regulated distinctly by innate stimuli vs signals from CD4+ T cells and reveal a process of trans activation of DC without secretion of polarizing cytokines that takes place during T cell priming and may be involved in amplifying immune responses.

Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting.

The protective effect of antibodies (Abs) is generally attributed to neutralization or complement activation. Using Legionella pneumophila and Mycobacterium bovis bacillus Calmette–Guérin as a model, we discovered an additional mechanism of Ab-mediated protection effective against intracellular pathogens that normally evade lysosomal fusion. We show that Fc receptor (FcR) engagement by Abs, which can be temporally and spatially separated from bacterial infection, renders the host cell nonpermissive for bacterial replication and targets the pathogens to lysosomes. This process is strictly dependent on kinases involved in FcR signaling but not on host cell protein synthesis or protease activation. Based on these findings, we propose a mechanism whereby Abs and FcR engagement subverts the strategies by which intracellular bacterial pathogens evade lysosomal degradation.

Induction and protective role of antibodies in Legionella pneumophila infection

Legionella pneumophila (Lpn) is a ubiquitous Gram‐negative bacterium found in aquatic environments and is the causative agent of Legionnaires’ disease, a severe form of pneumonia. We have used Lpn‐permissive A/J mice as a model to analyze the B cell response upon intravenous (i.v.) and intranasal (i.n.) infection with Lpn. A strong antibody (Ab) response was observed upon i.v. infection with wild‐type (WT) Lpn and an icmT mutant strain, which is unable to replicate within permissive host cells. In contrast to i.v. infection, only WT but not icmT mutant Lpn was able to induce specific Ab responses upon i.n. infection. After primary i.n. infection with WT Lpn, a strict compartmentalization of Lpn‐specific Ab isotypes was observed, as IgG was found exclusively systemically, while IgA was detectable only locally in the lung. Regardless of the infection route, isotype switching to IgG and to IgA was strictly dependent on CD4+ T cells, whereas IgM production was completely Th‐independent. Finally, we analyzed the protective capacity of the Lpn‐specific Ab response. Actively or passively immunized mice or mice that were infected with opsonized Lpn had 50–100‐fold reduced bacterial titers compared to naive animals, clearly demonstrating the capacity of Ab to protect against infection with Lpn.

Self peptide/MHC class I complexes have a negligible effect on the response of some CD8+ T cells to foreign antigen

MHC molecules loaded with self peptides do not trigger a T cell immune response but may deliver signals important for peripheral T cell survival and function. It is unclear if self peptide/MHCcomplexes on APC in addition can influence the T cell response to co‐presented foreign ligands. To address this question, TAP‐sufficient and TAP‐deficient cells were loaded with ovalbumin peptide (pOVA) to generate APC that present pOVA/H‐2Kb complexes in the context of high or low levels of self peptide‐loaded MHC class I, respectively. The two cell types were then used to stimulatedifferent CD8+ T cells specific for ovalbumin while the number of presented pOVA/H‐2Kb complexes was independently assessed by staining with 25‐D1, an antibody against pOVA/H‐2Kb. In each case, T cell activation was independent of TAP expression by the APC and depended exclusively on the amount of 25‐D1 staining. We conclude that the number of pOVA/Kb complexes and not their frequency relative to self peptide/MHC complexes determines the response of those T cells tested here. These results imply that the repertoire of self peptide/MHC class I complexes presented by APC has a negligible effect on the response of some CD8+ T cells to foreign ligands.